ArticleLiterature Review

Structure-function and biological role of betacellulin

September 2000
The International Journal of Biochemistry & Cell Biology 32(8):805-15

Source
PubMed

Authors:

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterised by a six-cysteine consensus motif that forms three intra-molecular disulfide bonds crucial for binding the ErbB receptor family. BTC was initially described, purified and cloned from a mouse insulinoma cell line. BTC is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. BTC binds and activates ErbB-1 and ErbB-4 homodimers and is further characterised by its unique ability to activate all possible heterodimeric ErbB receptors. BTC is widely expressed in most tissues and various body fluids, including milk. Expression is particularly high in the pancreas where it is thought to play a role in the differentiation of pancreatic beta cells. While much is known about the ErbB receptor binding characteristics of BTC and its effect on a variety of cultured cells under different conditions, the challenge that lies ahead is to determine the role of BTC in vivo. This review will focus on the structure of BTC and the various biological effects ascribed to this member of the EGF family.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

Article

Full-text available

May 2023

Background The gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined. Methods Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. Results The results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells. Conclusions BTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

Preprint

Full-text available

Jan 2023

Impaired Fertility in Transgenic Mice Overexpressing Betacellulin.

Article

Feb 2007

Ana A Gratao

Overlap between COPD genetic association results and transcriptional quantitative trait loci

Preprint

Jul 2024

Rationale: Genome-wide association studies (GWAS) have identified multiple genetic loci associated with chronic obstructive pulmonary disease (COPD). When integrated with GWAS results, expression quantitative trait locus (eQTL) studies can provide insight into biological mechanisms involved in disease by identifying single nucleotide polymorphisms (SNPs) that contribute to whole gene expression. However, there are multiple genetically driven regulatory and isoform-specific effects which cannot be detected in traditional eQTL analyses. Here, we identify SNPs that are associated with alternative splicing (sQTL) in addition to eQTLs to identify novel functions for COPD associated genetic variants. Methods: We performed RNA sequencing on whole blood from 3743 subjects in the COPDGene Study. RNA sequencing data from lung tissue of 1241 subjects from the Lung Tissue Research Consortium (LTRC), and whole genome sequencing data on all subjects. Associations between all SNPs within 1000 kb of a gene (cis-) and splice and gene expression quantifications were tested using tensorQTL. In COPDGene a total of 11,869,333 SNPs were tested for association with 58,318 splice clusters, and 8,792,206 SNPs were tested for association with 70,094 splice clusters in LTRC. We assessed colocalization with COPD-associated SNPs from a published GWAS[1]. Results After adjustment for multiple statistical testing, we identified 28,110 splice-sites corresponding to 3,889 unique genes that were significantly associated with genotype in COPDGene whole blood, and 58,258 splice-sites corresponding to 10,307 unique genes associated with genotype in LTRC lung tissue. We found 7,576 sQTL splice-sites corresponding to 2,110 sQTL genes were shared between whole blood and lung, while 20,534 sQTL splice-sites in 3,518 genes were unique to blood and 50,682 splice-sites in 9,677 genes were unique to lung. To determine what proportion of COPD-associated SNPs were associated with transcriptional splicing, we performed colocalization analysis between COPD GWAS and sQTL data, and found that 38 genomic windows, corresponding to 38 COPD GWAS loci had evidence of colocalization between QTLs and COPD. The top five colocalizations between COPD and lung sQTLs include NPNT, FBXO38, HHIP, NTN4 and BTC. Conclusions A total of 38 COPD GWAS loci contain evidence of sQTLs, suggesting that analysis of sQTLs in whole blood and lung tissue can provide novel insights into disease mechanisms.

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Article

Full-text available

Sep 2017

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2-Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4-Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC-pre-treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF-pre-treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.

Interstitial Outburst of Angiogenic Factors During Skeletal Muscle Regeneration After Acute Mechanical Trauma

Article

Aug 2015
ANAT REC

Angiogenesis is a key event during tissue regeneration, but the intimate mechanisms controlling this process are still largely unclear. Therefore, the cellular and molecular interplay along normal tissue regeneration should be carefully unveiled. To this matter, we investigated by xMAP assay the dynamics of some angiogenic factors known to be involved in tissue repair, such as follistatin (FST), Placental Growth Factor-2 (PLGF-2), epidermal growth factor (EGF), betacellulin (BTC) and amphiregulin (AREG) using an animal model that mimics acute muscle contusion injuries. In situ immunofluorescence was used for the evaluation and tissue distribution of their cellular sources. Tissue levels of explored factors increased significantly during degeneration and inflammatory stage of regeneration, peaking first week post-injury. However, except for PLGF-2 and EGF, their levels remained significantly elevated after the inflammatory process started to fade. Serum levels were significantly increased only after 24 h for AREG and EGF. Though, for all factors except FST, the levels in injured samples didn't correlate with serum or contralateral tissue levels, excluding the systemic influence. We found significant correlations between the levels of EGF and AREG, BTC, FST and FST and AREG in injured samples. Interstitial cells expressing these factors were highlighted by in situ immunolabelling and their number correlated with measured levels dynamics. Our study provides evidence of a dynamic level variation along the regeneration process and a potential interplay between selected angiogenic factors. They are synthesized, at least partially, by cell populations residing in skeletal muscle interstitium during regeneration after acute muscle trauma. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.

Trafficking of Epidermal Growth Factor Receptor Ligands in Polarized Epithelial Cells

Article

Nov 2013
ANNU REV PHYSIOL

A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes. Expected final online publication date for the Annual Review of Physiology Volume 76 is February 10, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Ligand-Dependent Activation of EGFR in Follicular Dendritic Cells Sarcoma is Sustained by Local Production of Cognate Ligands

Article

Full-text available

Jul 2013

Purpose: The aim of this study was to investigate the biological and clinical significance of epidermal growth factor receptor (EGFR) signaling pathway in follicular dendritic cell sarcoma (FDC-S). Experimental design: Expression of EGFR and cognate ligands as well as activation of EGFR signaling components was assessed in clinical samples and in a primary FDC-S short-term culture (referred as FDC-AM09). Biological effects of the EGFR antagonists cetuximab and panitumumab and the MEK inhibitor UO126 on FDC-S cells were determined in vitro on FDC-AM09. Direct sequencing of KRAS, BRAF, and PI3KCA was conducted on tumor DNA. Results: We found a strong EGFR expression on dysplastic and neoplastic FDCs. On FDC-AM09, we could show that engagement of surface EGFR by cognate ligands drives the survival and proliferation of FDC-S cells, by signaling to the nucleus mainly via MAPK and STAT pathways. Among EGFR ligands, heparin-binding EGF-like growth factor, TGF-α and Betacellulin (BTC) are produced in the tumor microenvironment of FDC-S at RNA level. By extending this finding at protein level we found that BTC is abundantly produced by FDC-S cells and surrounding stromal cells. Finally, direct sequencing of tumor-derived genomic DNA showed that mutations in KRAS, NRAS, BRAF, and PI3KCA, which predicts resistance to anti-EGFR MoAb in other cancer models, are not observed in FDC-S. Conclusion: Activation of EGFR by cognate ligands produced in the tumor microenvironment sustain viability and proliferation of FDC-S indicating that the receptor blockade might be clinically relevant in this neoplasm.

The epidermal growth factor receptor inhibitor gefitinib enhances in vitro and in vivo sensory axon regeneration and functional recovery following transection in a mouse median nerve injury model

Article

Full-text available

Nov 2024
MUSCLE NERVE

Introduction: The epidermal growth factor receptor (EGFR; ErbB1), a membrane bound receptor tyrosine kinase, is hypothesized to have an inhibitory influence on peripheral nerve regeneration. This study examines the impact of EGFR inhibition on nerve regeneration using the commercially available small molecule inhibitor gefitinib. Method: In vitro assays included neurite outgrowth of cultured dorsal root ganglion (DRG) neurons from adult C57Bl/6 wildtype mice on immobilized chondroitin sulfate proteoglycans (CSPG). Following forelimb median nerve injury, EGFR expression, number of regenerated neurons (using retrograde labeling) and myelination of motor and sensory neurons were compared between mice that received either gefitinib or vehicle. Functional recovery was assessed using grip strength. Results: EGFR expression on DRG and spinal motor neurons was confirmed. Gefitinib significantly increased neurite outgrowth in medium sized (30–50 μm) DRG neurons, resulting in longer neurites (183 ± 36 μm) compared with CSPG alone (49 ± 9 μm). After median nerve injury, significantly greater numbers of sensory neurons (638 ± 112 vs. 301 ± 81), but not motor neurons (31 ± 12 vs. 42 ± 13) regenerated in animals treated with gefitinib compared with controls. Regenerated axons in gefitinib treated animals displayed significantly greater diameter and increased g-ratiocompared with controls. Grip strength recovered more quickly in animals receiving gefitinib compared with controls (27.6 vs. 19.1 g 18 days post-injury). Discussion: This study provides data supporting the role of EGFR as a negative regulatorof sensory but not motor neuron regeneration. Further, it demonstrates versatile potential uses of existing pharmaceuticals

Accounting for dominance genetic variation in carcass quality traits of Canadian crossbred beef cattle

Article

Full-text available

Apr 2024

The objectives of this study were to evaluate the contribution of additive and dominance genetic effects to the phenotypic variation of carcass quality traits and to identify the underlying genetic variants associated with these traits. A total of 3958 Canadian crossbred beef cattle with phenotype and genotype data were used in two models: (1) additive and (2) joint additive and dominance genomic models that included fixed contemporary group, and covariates of slaughter age, and the eigenvectors of five principal components to account for population structure. Variance components and genome-wide association analyses were performed, and a 10% genome-wide false discovery rate (FDR) was applied to declare associations as significant. Genomic heritability ranged from 0.31 ± 0.03 for ultrasound rib eye area to 0.46 ± 0.05 for marbling score. Up to 10% dominance genetic variation was observed for ultrasound rib eye area and marbling score, indicating the contribution of dominance genetic effects to these trait variations. Eleven overlapping significant single-nucleotide polymorphism associations were identified across the studied traits and models. The identified candidate genes (e.g., BTC, SPP1, and SEPSECS) have biological functions related to tissue growth and skeletal muscle development and can be further validated in other cattle populations to determine their usefulness for beef cattle genetic improvement.

Functional attributes of bioactive peptides of bovine milk origin and application of in silico approaches for peptide prediction and functional annotations

Article

Full-text available

May 2023
CRIT REV FOOD SCI

Bovine milk peptides are the protein fragments with diverse bioactive properties having antioxidant, anticarcinogenic, other therapeutic and nutraceutical potentials. These peptides are formed in milk by enzymatic hydrolysis, gastrointestinal digestion and fermentation processes. They have significant health impact with high potency and low toxicity making them a suitable natural alternative for preventing and managing diseases. Antibiotic resistance has increased the quest for better peptide candidates with antimicrobial effects. This article presents a comprehensive review on well documented antimicrobial, immunological, opioid, and anti-hypertensive activities of bovine milk peptides. It also covers the usage of computational biology tools and databases for prediction and analysis of the food-derived bioactive peptides. In silico analysis of amino acid sequences of Bos taurus milk proteins have been predicted to generate peptides with dipeptidyl peptidase IV inhibitory and ACE inhibitory properties, making them favorable candidates for developing blood sugar lowering drugs and anti-hypertensives. In addition to the prediction of new bioactive peptides, application of bioinformatics tools to predict novel functions of already known peptides is also discussed. Overall, this review focuses on the reported as well as predicted biologically active peptide of casein and whey proteins of bovine milk that can be utilized to develop therapeutic agents.

Stem Cell Therapy in Combination with Naturopathy: Current Progressive Management of Diabetes and Associated Complications

Article

Dec 2022

Background Diabetes is a chronic metabolic disorder having a global prevalence of nearly doubled over the last 30 years and has become one of the major health concerns worldwide. The number of adults with diabetes increased to 537 million in 2021. Introduction The overarching goal of diabetic research and treatment has always been to restore insulin independence and an average blood glucose level. Chemotherapeutic antidiabetic agents can manage diabetes but often show toxicity and drug resistance. Natural phytomedicines may be useful along with stem cell therapy for diabetes management. Even if the whole pancreatic organ and islet transplantation, are becoming benchmark techniques for diabetes management and control, a considerable scarcity of eligible donors of pancreatic tissues and organs severely limits their use. Stem cell treatment provides a bunch of possibilities for treating people with diabetes. Methods For this purpose, comprehensive article searching was conducted, with relevant material obtained using search engines such as Scopus, PubMed, MEDLINE, Google, and others, using appropriate keywords. Results Stem cell therapies, including induced pluripotent stem cells and mesenchymal stem cells, are now becoming a popular area of investigation. Recent advancements in stem cell therapy might provide a feasible treatment option. Furthermore, in recent years, some novel bioactive compounds derived from plants have demonstrated antidiabetic action with higher potency than oral hypoglycaemic medications. Recent regenerative medicine and stem cell treatment advancements might subsequently provide a feasible diabetic management option. On the other hand, medicinal herbs have been considered a better choice for the extensive treatment of diabetes. Conclusion If proper attention is not given to control diabetes by antidiabetic chemotherapeutic agents, natural phytomedicine, and sophisticated treatment like stem cell therapy, then the lifespan of patients will be decreased, and some associated secondary problems will also arise. So, the present review attempts to discuss naturopathy as an alternative resource in combination with stem cell therapy for the progressive management of diabetes and associated disorders.

Betacellulin promotes tumor development and EGFR mutant lung cancer growth by stimulating the EGFR pathway and suppressing apoptosis

Article

Full-text available

Apr 2022

Oncogenic mutations in the EGFR gene account for 15-20% of lung adenocarcinoma (LUAD) cases. However, the mechanism for EGFR driven tumor development and growth is not fully understood. Here, using a mRNA expression profiling-based approach we identified betacellulin (BTC) as one the gene upregulated by oncogenic EGFR in a MAP kinase-dependent manner. BTC protein expression was markedly increased in LUAD patient samples compared to normal lung tissue, with higher expression in EGFR-mutant LUAD. BTC was sufficient to transform immortalized mouse cells, initiate tumor development in mice, and promote the survival of immortalized human lung epithelial cells. Conversely, knockdown of BTC inhibited the growth of EGFR-mutant human LUAD cells in culture and their tumor-forming ability in mice. Mechanistically, BTC knockdown resulted in attenuated EGFR signaling and apoptosis induction. Collectively, these results demonstrate a key role of BTC in EGFR-mutant LUAD, with potential therapeutic implications in LUAD and other EGFR-mutant cancers.

The Yin and Yang of ERBB4: Tumor Suppressor and Oncoprotein

Article

Full-text available

Jan 2022

ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are oncoproteins and validated targets for therapeutic intervention in a variety of solid tumors. In contrast, the role that ERBB4 plays in human malignancies is ambiguous. Thus, here we review the literature regarding ERBB4 function in human malignancies. We review the mechanisms of ERBB4 signaling with an emphasis on mechanisms of signaling specificity. In the context of this signaling specificity, we discuss the hypothesis that ERBB4 appears to function as a tumor suppressor protein and as an oncoprotein. Next, we review the literature that describes the role of ERBB4 in tumors of the bladder, liver, prostate, brain, colon, stomach, lung, bone, ovary, thyroid, hematopoietic tissues, pancreas, breast, skin, head, and neck. Whenever possible, we discuss the possibility that ERBB4 mutants function as biomarkers in these tumors. Finally, we discuss the potential roles of ERBB4 mutants in the staging of human tumors and how ERBB4 function may dictate the treatment of human tumors. SIGNIFICANCE STATEMENT: This articles reviews ERBB4 function in the context of the mechanistic model that ERBB4 homodimers function as tumor suppressors, whereas ERBB4-EGFR or ERBB4-ERBB2 heterodimers act as oncogenes. Thus, this review serves as a mechanistic framework for clinicians and scientists to consider the role of ERBB4 and ERBB4 mutants in staging and treating human tumors.

Genetics Basis of Gonadotropin-Releasing Hormone GnRH

Book

Mar 2020

This book systematically introduces the basic knowledge of the Hypothalamic Pituitary Gonads (HPG) axis and provides information on the location and regulation, gene mutations, function, reproduction, life cycle, sexual behaviour, disorders and the role of the environmental factors on GnRH gene. Also, we focused on gene and receptor structures, and the signalling pathways of GnRH, and its related genes and hormones such as Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Progesterone (P4) and Oestradiol (E2). Also, it was pointed to Gonadotropin Inhibiting Hormone (GnIH) and its related peptides, such as RFamide peptides which were found to decrease hormones secretion by working on HPG in an inhibiting biosynthesis process of gonadotropin (α–β) subunits. In addition, the roles of hormones on fertility and reproduction, also, disruption resulted from mutations. Special characteristics of many hormones and pulsatile secretions of GnRH were summarized.

Inhibition of Brain EGFR Activation: A Novel Target in Neurodegenerative Diseases and Brain Injuries

Article

Full-text available

Apr 2020

Several reports have been published recently demonstrating a beneficial effect of epidermal growth factor receptor (EGFR) inhibitors in improving pathological and behavioral conditions in neurodegenerative diseases (NDDs) such as Alzheimer’s disease (AD) and Amyotrophic Lateral Sclerosis (ALS) as well as the brain and spinal cord injuries (SCI). Despite successful therapeutics effects of EGFR inhibition in these pathological conditions, there is still no report of proof-of-concept studies in well-characterized animal models using recently developed Blood- Brain-Barrier (BBB) penetrating EGFR inhibitors which is due to previous conflicting reports concerning the level of EGFR or activated EGFR in normal and pathological conditions which caused target engagement a concern in any future EGFR inhibition therapy. In this review, the level of EGFR expression and activation in developing CNS compared to adult CNS will be explained as well as how neuronal injury or pathological conditions especially inflammation and amyloid fibrils induce reactive astrocytes leading to increase in the expression and activation of EGFR and finally neurodegeneration. Furthermore, in this review, we will discuss two main molecular mechanisms that can be proposed as neuroprotective effects of EGFR inhibition in these pathological conditions. We will also review the recent advances in the development of BBB- penetrating EGFR inhibitors in cancer therapy, which may eventually be repositioned for NDDs and SCI therapy in the future.

Betacellulin enhances ovarian cancer cell migration by up-regulating Connexin43 via MEK-ERK signaling

Article

Oct 2019
CELL SIGNAL

Gene therapy and type 1 diabetes mellitus

Article

Full-text available

Dec 2018
BIOMED PHARMACOTHER

Background Type 1 diabetes mellitus (T1DM) is an autoimmune disorder characterized by T cell-mediated self-destruction of insulin-secreting islet β cells. Management of T1DM is challenging and complicated especially with conventional medications. Gene therapy has emerged as one of the potential therapeutic alternatives to treat T1DM. This review primarily focuses on the current status and the future perspectives of gene therapy in the management of T1DM. A vast number of the studies which are reported on gene therapy for the management of T1DM are done in animal models and in preclinical studies. In addition, the safety of such therapies is yet to be established in humans. Currently, there are several gene level interventions that are being investigated, notably, overexpression of genes and proteins needed against T1DM, transplantation of cells that express the genes against T1DM, stem-cells mediated gene therapy, genetic vaccination, immunological precursor cell-mediated gene therapy and vectors. Methods We searched the current literature through searchable online databases, journals and other library sources using relevant keywords and search parameters. Only relevant publications in English, between the years 2000 and 2018, with evidences and proper citations, were considered. The publications were then analyzed and segregated into several subtopics based on common words and content. A total of 126 studies were found suitable for this review. Findings Generally, the pros and cons of each of the gene-based therapies have been discussed based on the results collected from the literature. However, there are certain interventions that require further detailed studies to ensure their effectiveness. We have also highlighted the future direction and perspectives in gene therapy, which, researchers could benefit from.

Betacellulin regulates schwann cell proliferation and myelin formation in the injured mouse peripheral nerve

Article

Jan 2017
GLIA

When a nerve fiber is cut or crushed, the axon segment that is separated from the soma degenerates distal from the injury in a process termed Wallerian degeneration (WD). C57BL/6OlaHsd-Wld(S) (Wld(S) ) mutant mice exhibit significant delays in WD. This results in considerably delayed Schwann cell and macrophage responses and thus in impaired nerve regenerations. In our previous work, thousands of genes were screened by DNA microarrays and over 700 transcripts were found to be differentially expressed in the injured sciatic nerve of Wld(S) compared with wild-type (WT) mice. One of these transcripts, betacellulin (Btc), was selected for further analysis since it has yet to be characterized in the nervous system, despite being known as a ligand of the ErbB receptor family. We show that Btc mRNA is strongly upregulated in immature and dedifferentiated Sox2(+) Schwann cells located in the sciatic nerve distal stump of WT mice, but not Wld(S) mutants. Transgenic mice ubiquitously overexpressing Btc (Tg-Btc) have increased numbers of Schmidt-Lantermann incisures compared with WT mice, as revealed by Coherent anti-Stokes Raman scattering (CARS). Tg-Btc mice also have faster nerve conduction velocity. Finally, we found that deficiency in Btc reduces the proliferation of myelinating Schwann cells after sciatic nerve injury, while Btc overexpression induces Schwann cell proliferation and improves recovery of locomotor function. Taken together, these results suggest a novel regulatory role of Btc in axon-Schwann cell interactions involved in myelin formation and nerve repair. GLIA 2017.

The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Article

Full-text available

Dec 2016

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies.

Milk Minor Constituents, Enzymes, Hormones, Growth Factors, and Organic Acids

Article

Apr 2013

Lígia R Rodrigues

Milk and derived products contain essential nutrients, such as proteins, lactose, minerals, vitamins, and enzymes. Additionally, despite their low concentrations in milk, many other minor constituents have important physiological and/or technological roles (e.g. hormones, growth factors). The dairy industry faces many challenges regarding milk processing. Also, knowledge of these constituents' physiological roles and effects on health is still lacking. Technological advances, innovative research approaches using metabolic engineering, systems and synthetic biology, and novel production methods will allow the production of higher amounts of such constituents, so that their recovery is easier. This will allow the production of differentiated compounds, thus revolutionizing the fields of personalized nutrition and functional foods. This chapter focuses on the description of the minor milk constituents, their applications and main technological challenges. Future perspectives and concerns related to these constituents are discussed.

4q13.3 - Amplifikationen in humanen Tumoren

Article

Nov 2011

Mareike Julia Sandmann

Role of epidermal growth factor receptor in the pathogenesis and treatment of arterial hypertension

Article

Feb 2007

Epidermal growth factor receptor (EGFR) is a member of receptor tyrosine kinase family. Upon ligand binding, EGFR undergoes autophosphorylation at several tyrosine residues within its intracellular domain and triggers a number of signaling pathways including extracellular signal-regulated kinases, phospholipase C?, and phosphoinositide 3-kinase. EGFR regulates cell growth, proliferation and survival, and its overexpression or oncogenic mutations are observed in many human cancers. Therefore, several drugs have been developed to target EGFR for antitumor therapy. In the cardiovascular system, enhanced EGFR signaling is involved in vascular and myocardial hypertrophy. Recent studies suggest that EGFR may contribute to the development of arterial hypertension. Stimulated EGFR induces vasoconstriction and regulates renal tubular Na+ transport. Apart from its cognate ligand, EGFR is activated by many vasoconstrictors including angiotensin II, norepinephrine and endothelin-1. Enhanced EGFR signaling has been observed in several animal models of hypertension, and synthetic EGFR inhibitors reduce blood pressure in some of these models. Leptin, a peptide hormone which is produced by white adipose tissue and circulates in increased amounts in obese subjects, transactivates EGFR in vascular wall and the kidney, and EGFR inhibitor, AG1478, reduces blood pressure in experimental hyperleptinemia, suggesting that leptin-induced activation of EGFR may be involved in the pathogenesis of hypertension associated with the metabolic syndrome. These data suggest that inhibiting EGFR could be a novel therapeutic strategy for the treatment of hypertension. In addition, some of currently used hypotensive medications, in particular inhibitors of the renin-angiotensin-aldosterone system, modulate EGFR signaling.

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

Article

Full-text available

Dec 2014
TISSUE CELL

Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. The aim of this study was to evaluate FG scaffold effect on differentiation of insulin-producing cells (IPCs) from bone marrow-derived mesenchymal stem cells (BM-MSCs). In this experimental study BM-MSCs were cultured and the cells characterized by analysis of cell surface markers using flow cytometry. BM-MSCs were seeded in FG scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was demonstrated using gene expression profiles for pancreatic cell differentiation markers (PDX-1, GLUT-2 and insulin) and insulin detection in cytoplasm. Release of insulin by these cells was confirmed by radioimmunoassay. Expression of the islet-associated genes PDX-1, GLUT-2 and Insulin genes in 3D cultured cells was markedly higher than the 2D cultured cells exposure differentiation media. Compared to 2D culture of BM-MSCs-derived IPCs, the insulin release from 3D BM-MSCs-derived IPCs showed a nearly 3 fold (p<0.05) increase when exposed to a high glucose (25mM) medium. Percentage of insulin positive cells in 3D experimental group showed an approximately 3.5-fold increase in compared to 2D experimental culture cells. The results of this study demonstrated that FG scaffold can enhance the differentiation of IPCs from rats BM-MSCs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

Article

Full-text available

Dec 2014

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.

Type II cGMP-dependent protein kinase inhibits ligand-induced activation of EGFR in gastric cancer cells

Article

Full-text available

Feb 2014
Mol Med Rep

Our previous data demonstrated that type II cGMP‑dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)-induced MAPK/ERK/JNK‑mediated signal transduction through inhibiting the phosphorylation/activation of the epidermal growth factor receptor (EGFR). Since the EGFR also binds with several other ligands as well as EGF, the present study was designed to investigate whether PKG II inhibited transforming growth factor-α (TGF-α), betacellulin (BTC) and epiregulin (EPR) induced phosphorylation/activation of the EGFR and consequent MAPK/ERK‑mediated signaling. The human gastric cancer cell line AGS, was infected with adenoviral constructs encoding cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II and was treated with 8-pCPT‑cGMP to activate the kinase. Western blotting was applied to detect the phosphorylation of EGFR and MAPK/ERK. The results demonstrated that treatment with EGF (100 ng/ml, 5 min), TGF-α (100 ng/ml, 5 min), BTC (100 ng/ml, 5 min) and EPR (100 ng/ml, 5 min) increased the tyrosine (tyr) 1068 phosphorylation of the EGFR and the threonine (thr) 202/tyr 204 phosphorylation of MAPK/ERK. Infecting the cells with Ad-PKG II and stimulating the kinase with 8-pCPT-cGMP efficiently inhibited the phosphorylation of the EGFR and MAPK/ERK induced by EGF, TGF-α, BTC and EPR. The results indicated that PKG II also inhibits the activation of the EGFR caused by diverse ligands of the receptor.

A systems biology approach using metabolomic data reveals genes and pathways interacting to modulate divergent growth in cattle

Article

Full-text available

Nov 2013
BMC GENOMICS

Systems biology enables the identification of gene networks that modulate complex traits. Comprehensive metabolomic analyses provide innovative phenotypes that are intermediate between the initiator of genetic variability, the genome, and raw phenotypes that are influenced by a large number of environmental effects. The present study combines two concepts, systems biology and metabolic analyses, in an approach without prior functional hypothesis in order to dissect genes and molecular pathways that modulate differential growth at the onset of puberty in male cattle. Furthermore, this integrative strategy was applied to specifically explore distinctive gene interactions of non-SMC condensin I complex, subunit G (NCAPG) and myostatin (GDF8), known modulators of pre- and postnatal growth that are only partially understood for their molecular pathways affecting differential body weight. Our study successfully established gene networks and interacting partners affecting growth at the onset of puberty in cattle. We demonstrated the biological relevance of the created networks by comparison to randomly created networks. Our data showed that GnRH (Gonadotropin-releasing hormone) signaling is associated with divergent growth at the onset of puberty and revealed two highly connected hubs, BTC and DGKH, within the network. Both genes are known to directly interact with the GnRH signaling pathway. Furthermore, a gene interaction network for NCAPG containing 14 densely connected genes revealed novel information concerning the functional role of NCAPG in divergent growth. Merging both concepts, systems biology and metabolomic analyses, successfully yielded new insights into gene networks and interacting partners affecting growth at the onset of puberty in cattle. Genetic modulation in GnRH signaling was identified as key modifier of differential cattle growth at the onset of puberty. In addition, the benefit of our innovative concept without prior functional hypothesis was demonstrated by data suggesting that NCAPG might contribute to vascular smooth muscle contraction by indirect effects on the NO pathway via modulation of arginine metabolism. Our study shows for the first time in cattle that integration of genetic, physiological and metabolomics data in a systems biology approach will enable (or contribute to) an improved understanding of metabolic and gene networks and genotype-phenotype relationships.

Endothelial Cells Promote Pseudo-islet Function Through BTC-EGFR-JAK/STAT Signaling Pathways

Article

Jun 2024
ANN BIOMED ENG

Interactions between cells are of fundamental importance in affecting cell function. In vivo, endothelial cells and islet cells are close to each other, which makes endothelial cells essential for islet cell development and maintenance of islet cell function. We used endothelial cells to construct 3D pseudo-islets, which demonstrated better glucose regulation and greater insulin secretion compared to conventional pseudo-islets in both in vivo and in vitro trials. However, the underlying mechanism of how endothelial cells promote beta cell function localized within islets is still unknown. We performed transcriptomic sequencing, differential gene analysis, and enrichment analysis on two types of pseudo-islets to show that endothelial cells can promote the function of internal beta cells in pseudo-islets through the BTC-EGFR-JAK/STAT signaling pathway. Min6 cells secreted additional BTC after co-culture of endothelial cells with MIN6 cells outside the body. After BTC knockout in vitro, we found that beta cells functioned differently: insulin secretion levels decreased significantly, while the expression of key proteins in the EGFR-mediated JAK/STAT signaling pathway simultaneously decreased, further confirming our results. Through our experiments, we elucidate the molecular mechanisms by which endothelial cells maintain islet function in vitro, which provides a theoretical basis for the construction of pseudo-islets and islet cell transplants for the treatment of diabetes mellitus.

Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

Article

Full-text available

Mar 2024
eLife

Human Epidermal growth factor Receptor 4 (HER4 or ERBB4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1β. Here, we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1β and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1β and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers.

Targeted Inhibitors of EGFR: Structure, Biology, Biomarkers, and Clinical Applications

Article

Full-text available

Dec 2023

Members of the EGFR family of tyrosine kinase receptors are major regulators of cellular proliferation, differentiation, and survival. In humans, abnormal activation of EGFR is associated with the development and progression of many cancer types, which makes it an attractive target for molecular-guided therapy. Two classes of EGFR-targeted cancer therapeutics include monoclonal antibodies (mAbs), which bind to the extracellular domain of EGFR, and tyrosine kinase inhibitors (TKIs), which mostly target the intracellular part of EGFR and inhibit its activity in molecular signaling. While EGFR-specific mAbs and three generations of TKIs have demonstrated clinical efficacy in various settings, molecular evolution of tumors leads to apparent and sometimes inevitable resistance to current therapeutics, which highlights the need for deeper research in this field. Here, we tried to provide a comprehensive and systematic overview of the rationale, molecular mechanisms, and clinical significance of the current EGFR-targeting drugs, highlighting potential candidate molecules in development. We summarized the underlying mechanisms of resistance and available personalized predictive approaches that may lead to improved efficacy of EGFR-targeted therapies. We also discuss recent developments and the use of specific therapeutic strategies, such as multi-targeting agents and combination therapies, for overcoming cancer resistance to EGFR-specific drugs.

Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

Preprint

Nov 2023
eLife

Human Epidermal growth factor Receptor 4 (HER4) carries out essential functions in the development and maintenance of the cardiovascular and nervous systems. HER4 activation is regulated by a diverse group of extracellular ligands including the neuregulin (NRG) family and betacellulin (BTC), which promote HER4 homodimerization or heterodimerization with other HER receptors. Important cardiovascular functions of HER4 are exerted via heterodimerization with its close homolog and orphan receptor, HER2. To date structural insights into ligand-mediated HER4 activation have been limited to crystallographic studies of HER4 ectodomain homodimers in complex with NRG1β. Here we report cryo-EM structures of near full-length HER2/HER4 heterodimers and full-length HER4 homodimers bound to NRG1β and BTC. We show that the structures of the heterodimers bound to either ligand are nearly identical and that in both cases the HER2/HER4 heterodimer interface is less dynamic than those observed in structures of HER2/EGFR and HER2/HER3 heterodimers. In contrast, structures of full-length HER4 homodimers bound to NRG1β and BTC display more large-scale dynamics mirroring states previously reported for EGFR homodimers. Our structures also reveal the presence of multiple glycan modifications within HER4 ectodomains, modeled for the first time in HER receptors, that distinctively contribute to the stabilization of HER4 homodimer interfaces over those of HER2/HER4 heterodimers.

Structural dynamics of the active HER4 and HER2/HER4 complexes is finely tuned by different growth factors and glycosylation

Preprint

Full-text available

Nov 2023

Cloning, expression and purification of epidermal growth factor receptor for aptamer selection for certain bioassays

Thesis

Full-text available

Jan 2022

Sambhavi Animesh

The present study demonstrates the selection and characterization of DNA aptamers specific to the extra cellular domain (ECD) of Epidermal Growth Factor Receptor (EGFR) protein. In this pursuit, the ECD of EGFR was cloned, expressed and purified and a panel of DNA aptamers binding specifically to EGFR protein was selected by Systematic Evolution of Ligands by EXponential enrichment (SELEX). Then the candidate aptamers were sequenced and their potential secondary structure was predicted by mfold software. The binding affinities of the selected aptamers were determined by Flow cytometry and ELISA. The application of selected aptamers in various bioassays were explored. For example the selected aptamers were used as a bioimaging probe for the detection of EGFR overexpression in cancer cell lines and as a detection probe in membrane based assay like Dot blot assay. In vitro cell culture model was employed to ascertain the anti-cytotoxicity and anti-migratory effect of the selected aptamers. Further the aptamer with best recognition ability was used for the development of microtiter based assay for the detection of EGFR protein.

HPP1 Ectodomain Shedding is Mediated by ADAM17 and is Necessary for Tumor Suppression in Colon Cancer

Article

Oct 2020

Background Hyperplastic polyposis protein 1 (HPP1) encodes a tumor-suppressive transmembrane cleavable epidermal growth factor–like ligand. It is unclear as to whether cleavage and shedding of HPP1 are essential steps in achieving its tumor suppressive properties. ADAM proteins are key players in cellular ectodomain shedding processes with ADAM17 being well characterized and representing the most likely sheddase for HPP1. In this study, we explore the mechanisms and importance of ectodomain shedding in contributing to HPP1-mediated tumor suppression. Methods Baseline characterization of HPP1 ectodomain shedding and ADAM family member expression was performed in HCT116 colon cancer cells with forced overexpression of HPP1 and controls. Subsequent impact of attenuation of ADAM expression by short interfering RNA on HPP1 shedding was evaluated. Furthermore, we examined the functional impact of an uncleavable HPP1 mutant construct (HPP1-Δstalk) generated by site-directed mutagenesis. Cellular growth potential functions were analyzed by MTT and soft agar assays. Results Select proinflammatory cytokines enhanced HPP1 ectodomain shedding, whereas short interfering RNA–mediated knockdown of ADAM17 resulted in abrogation of HPP1 ectodomain shedding. ADAM17 knockdown concomitantly resulted in increased cell proliferation and anchorage-independent growth. HPP1-Δstalk–transfected cells exhibited significantly higher proliferation and reduced STAT1 activation relative to full-length HPP1, further suggesting a critical role for ectodomain shedding in HPP1-mediated tumor suppression. Conclusion The tumor-suppressive properties of HPP1 in colorectal cancer require cleavage and shedding of its ectodomain which in turn are mediated by ADAM17. Further investigations into the regulation of HPP1 may lead to a greater understanding of epidermal growth factor–like ligand family biology and potential novel therapeutic strategies.

Biomaterials and controlled release strategy for epithelial wound healing

Article

Jul 2019

Skin and cornea are tissues providing protecting functions. Trauma and other environmental threats often cause injuries, infections and damages of the tissues. The degree of injury will directly correlate to the length of recovery time. For example, a superficial skin or corneal wound may recover within days; however, more severe injuries can last up to several months and may leave scarring. Therapeutic strategies have been introduced to enhance wound healing efficiency and quality. Although skin and cornea share similar anatomic structures and wound healing process, therapeutic agents and formulations for skin and cornea wound healing differ in accordance with tissue and wound types. In this review, we describe the anatomy and epithelial wound healing processes of skin and cornea, and summarize the therapeutic molecules that are beneficial to the respective regeneration process. In addition, biomaterial scaffolds that inherently possess bioactive properties or modified with therapeutic molecules for topical controlled release and enhanced wound healing efficiency are also discussed.

Loss of RHBDF2 results in an early-onset spontaneous murine colitis

Article

Full-text available

Jan 2019

Inflammatory bowel disease (IBD) is a heterogeneous group of inflammation‐mediated pathologies that include Crohn's disease and ulcerative colitis and primarily affects the colon and small intestine. Previous studies have shown that a disintegrin and metalloprotease (ADAM) 17, a membrane‐bound sheddase, capable of cleaving the proinflammatory cytokine TNF and epidermal growth factor receptor ligands, plays a critical role in maintaining gut homeostasis and modulating intestinal inflammation during IBD. Rhomboid 5 homolog 2 (RHBDF2), a catalytically inactive member of the rhomboid family of intramembrane serine proteases, was recently identified as a crucial regulator of ADAM17. Here, we assessed the role of RHBDF2 in the development of colitis in the context of IL10 deficiency. Il10−/−/Rhbdf2−/− mice developed spontaneous colitis and experienced severe weight loss starting at 8 wk of age, without the need for exogenous triggers. Severity of disease pathology in Il10−/−/Rhbdf2−/− mice correlated with a dysbiotic gut microbiota and elevated Th1‐associated immune responses with increased interferon gamma and IL2 production. In addition, Il10−/−/Rhbdf2−/− mice failed to maintain their epithelial cell homeostasis, although the intestinal epithelial barrier of Rhbdf2−/− mice is intact and loss of Rhbdf2 did not significantly exacerbate sensitivity to dextran sulfate sodium‐induced colitis, suggesting differences in the underlying disease pathway of intestinal inflammation in this model. Taken together, our results demonstrate a critical regulatory role for RHBDF2 in the maintenance of the unique homeostasis between intestinal microbiota and host immune responses in the gut that is dysregulated during the pathogenesis of IBD. RHBDF2 participates in maintaining the critical balance between intestinal microbiota and host immune responses during the pathogenesis of colitis in Il10‐deficient mice.

β-cellulin promotes the proliferation of corneal epithelial stem cells through the phosphorylation of erk1/2

Article

Jan 2018

The proliferation of corneal epithelial stem cells (CESCs) is a very important process in the recovery of corneal wounds. Recent studies have shown that β-cellulin (BC) is effective in the repair of other tissues. However, its mechanism of action in corneal wound healing is not yet clear. The purpose of this study was to investigate how BC accelerates wound healing of the cornea. Here, we confirmed that the proliferation of CESCs was induced at a specific concentration (0.2, 2 and 20 ng/mL) by treatment with BC. Markers associated with proliferation activity (ΔNp63, bmi-1, abcg2) were also upregulated. In vivo experiments showed that the corneal wound healing rate was increased in mice. We found that BC stimulates the phosphorylation of the erk1/2 signaling pathway, which is triggered during the recovery of mouse corneal wounds. However, the inhibition of erk1/2 phosphorylation delayed the recovery of mouse corneal wounds in an organ culture assay. According to these results, BC may be a potential treatment factor for corneal wound healing.

Tumor-linked HER2 expression: Association with obesity and lipid-related microenvironment

Article

Oct 2017
Horm Mol Biol Clin Investig

Amitabha Ray

Obesity is associated with the risk of several health disorders including certain cancers. Among obesity-related cancers, postmenopausal breast carcinoma is a well-studied one. Apart from an increase in certain types of lipids in obesity, excess adipose tissue releases many hormone-like cytokines/adipokines, which are usually pro-inflammatory in nature. Leptin is one of such adipokines and significantly linked with the intracellular signaling pathways of other growth factors such as insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), human epidermal growth factor receptor 2 (HER2). In general, HER2 is overexpressed in roughly 30% of breast carcinomas; its presence indicates aggressive tumor behavior. Conversely, HER2 has certain effects in normal conditions such as differentiation of preadipocytes, cardiovascular health and vitamin D metabolism. HER2 has no known endogenous ligand, but it may form dimers with other three members of the epidermal growth factor receptor (EGFR) family and can activate downstream signaling pathways. Furthermore, HER2 is intimately connected with several enzymes, e.g. fatty acid synthase (FASN), phosphatidylinositol 3-kinase (PI3K), AKT and mechanistic target of rapamycin (mTOR), all of which play significant regulatory roles in lipogenic pathways or lipid metabolism. In obesity-related carcinogenesis, characteristics like insulin resistance and elevated IGF-1 are commonly observed. Both IGF-1 and leptin can modulate EGFR and HER2 signaling pathways. Although clinical studies have shown mixed results, the behavior of HER2+ tumor cells including HER2 levels can be altered by several factors such as obesity, leptin and fatty acids. A precise knowledge is useful in new therapeutic approaches against HER+ tumors.

ETS transcription factor ELF5 induces lumen formation in a 3D model of mammary morphogenesis and its expression is inhibited by Jak2 inhibitor TG101348

Article

Aug 2017
EXP CELL RES

The loss of expression of a single gene can revert normal tissue to a malignant phenotype. For example, while normal breast has high lumenal expression of CEACAM1, the majority of breast cancers exhibit the early loss of this gene with the concurrent loss of their lumenal phenotype. MCF7 cells that lack CEACAM1 expression and fail to form lumena in 3D culture, regain the normal phenotype when transfected with CEACAM1. In order to probe the mechanism of this gain of function, we treated these cells with the clinically relevant Jak2 inhibitor TG101348 (TG), expecting that disruption of the prolactin receptor signaling pathway would interfere with the positive effects of transfection of MCF7 cells with CEACAM1. Indeed, lumen formation was inhibited, resulting in the down regulation of a set of genes, likely involved in the complex process of lumen formation. As expected, inhibition of the expression of many of these genes also inhibited lumen formation, confirming their involvement in a single pathway. Among the genes identified by the inhibition assay, ETS transcription factor ELF5 stood out, since it has been identified as a master regulator of mammary morphogenesis, and is associated with prolactin receptor signaling. When ELF5 was transfected into the parental MCF7 cells that lack CEACAM1, lumen formation was restored, indicating that ELF5 can replace CEACAM1 in this model system of lumenogenesis. We conclude that the event(s) that led to the loss of expression of CEACAM1 is epistatic in that multiple genes associated with a critical pathway were affected, but that restoration of the normal phenotype can be achieved with reactivation of certain genes at various nodal points in tissue morphogenesis.

Epidermal Growth Factor-like Ligands

Chapter

Full-text available

Jan 2014

Growth Factors and Their Receptors in Cell Differentiation, Cancer and Cancer Therapy

Book

Jan 2011

Gajanan V Sherbet

Nuclear Energy is one of the most popular texts ever published on basic nuclear physics, systems, and applications of nuclear energy. This newest edition continues the tradition of offering a holistic treatment of everything the undergraduate engineering student needs to know in a clear and accessible way. Presented is a comprehensive overview of radioactivity, radiation protection, nuclear reactors, waste disposal, and nuclear medicine. New coverage on nuclear safety concerns following 9/11, including radiation and terrorism, nuclear plant security, and use of nuclear techniques to detect weapons materials. New facts on nuclear waste management, including the Yucca Mountain repository. New developments in the use of nuclear-powered systems for generating cheap and abundant hydrogen from water using nuclear technology. New information on prospects for new nuclear power reactors and their applications for electricity and desalination. New end-of-chapter Exercises and Answers, lists of Internet resources, and updated references. New instructor web site including Solutions to Exercises and PowerPoint slides. New student web site containing computer programs for use with Computer Exercises.

Extracellular signals and pancreatic beta-cell development: A brief review

Article

Dec 2002

Cell lineage development is a finely tuned process of proliferation and differentiation, survival and apoptosis, that is regulated by numerous extracellular signals. Here we review some of the extracellular signals-including insoluble cell-cell and extracellular matrix-cell interactions, as well as soluble factors-that appear critical for pancreatic beta-cell development. Knowledge of how these signals control the development of pancreatic endocrine stem/precursor cells into fully functional insulin-secreting beta cells is a platform for the restoration of beta-cell function and the cure therapy of type 1 diabetes.

Recent Advances in In Vivo Regeneration of Beta Cells by Growth Factors

Article

Mar 2010

Both type 1 and type 2 diabetes are characterized by β cell loss and dysfunction. Therefore, replenishment of functional beta cells by either transplantation, or in vivo β cell regeneration is a therapeutic option. β cell mass can be increased by promoting replication of existing cells, differentiation from stem/progenitor cells and inhibiting beta cell apoptosis. In this review, we will summarize the recent advances in in vivo regeneration of insulin- producing β cells with various growth factors such as betacellulin, glucagon-like peptide-1, gastrin, hepatocyte growth factor and prolactin.

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

Article

Sep 2015

The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p < 0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.

Ovulation

Chapter

Dec 2006

This chapter provides an overview of ovulation-a complicated path of molecular events initiated when luteinizing hormone (LH) couples with LH/human chorionic gonadotropin (hCG) receptors located in the membranes of granulosa cells as well as theca cells of mature ovarian follicles. These G-protein-coupled receptors operate through cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathways to induce the expression of a repertoire of genes in granulosa cells that are associated with the up-regulation of inflammatory cytokines such as IL-1β, IL-6, and TNF-α. Within this inflammatory blitz of up-regulated gene expression is the inducible form of cyclooxygenase, namely COX-2, which catalyzes the conversion of arachidonic acid into several prostanoids including the vasodilatory prostaglandin E2. One known, critical site of prostaglandin E2 (PGE2) action occurs during cell-enclosed oocyte complex (COC) expansion where this prostaglandin induces TSG-6, a factor that is obligatory for stabilizing the extracellular hyaluronan-rich COC matrix. A possible additional consequence of the biological action of prostaglandins is the hyperemic response that occurs during the first hours of the ovulatory process.

Betacellulin ameliorates hyperglycemia in obese diabetic db/db mice

Article

Jun 2015
J MOL MED-JMM

We found that administration of a recombinant adenovirus (rAd) expressing betacellulin (BTC) into obese diabetic db/db mice ameliorated hyperglycemia. Exogenous glucose clearance was significantly improved, and serum insulin levels were significantly higher in rAd-BTC-treated mice than rAd-β-gal-treated control mice. rAd-BTC treatment increased insulin/bromodeoxyuridine double-positive cells in the islets, and islets from rAd-BTC-treated mice exhibited a significant increase in the level of G1-S phase-related cyclins as compared with control mice. In addition, BTC treatment increased messenger RNA (mRNA) and protein levels of these cyclins and cyclin-dependent kinases in MIN-6 cells. BTC treatment induced intracellular Ca(2+) levels through phospholipase C-γ1 activation, and upregulated calcineurin B (CnB1) levels as well as calcineurin activity. Upregulation of CnB1 by BTC treatment was observed in isolated islet cells from db/db mice. When treated with CnB1 small interfering RNA (siRNA) in MIN-6 cells and isolated islets, induction of cell cycle regulators by BTC treatment was blocked and consequently reduced BTC-induced cell viability. As well as BTC's effects on cell survival and insulin secretion, our findings demonstrate a novel pathway by which BTC controls beta-cell regeneration in the obese diabetic condition by regulating G1-S phase cell cycle expression through Ca(2+) signaling pathways. Administration of BTC to db/db mice results in amelioration of hyperglycemia. BTC stimulates beta-cell proliferation in db/db mice. Ca(2+) signaling was involved in BTC-induced beta-cell proliferation. BTC has an anti-apoptotic effect and potentiates glucose-stimulated insulin secretion.

Studies on Memo, an important ErbB2 receptor-mediated component of the cellular migratory machinery

Article

Jan 2009

Maria Meira

Identifying Disease-Related Genes by Topological Features of Protein-Protein Interaction Network

Article

Dec 2011

To help the biomedical scientist pre-confirm the disease-related genes, we considered these gene as a whole research set and analyzed the topological features of their interaction network. Two strategies had been proposed to construct the disease-related gene network from the OMIM database. Using these two constructed sets, we trained two support vector machine prediction models, the accuracy of which are 75.09% and 83.63%. As a result, we gained 27 and 2873 potential disease-related genes respectively. The intersection of the two predicted sets contains 19 genes. In addition, gene locus's with high appearance frequency were listed for further research.

Prolidase Directly Binds and Activates Epidermal Growth Factor Receptor and Stimulates Downstream Signaling

Article

Full-text available

Dec 2012
J BIOL CHEM

Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation. Background: All known ligands of EGF receptor (EGFR) are characterized by the EGF motif and generated from transmembrane precursors. Results: Prolidase, a cytosolic dipeptidase devoid of EGF motif, binds and activates EGFR independent of its dipeptidase activity when present outside of cell. Conclusion: Prolidase is a novel EGFR ligand. Significance: This shows a new function of prolidase and new mechanism of EGFR activation.

Yoshida M, Shimura T, Sato M, Ebi M, Nakazawa T, Takeyama H, Joh TA novel predictive strategy by immunohistochemical analysis of four EGFR ligands in metastatic colorectal cancer treated with anti-EGFR antibodies. J Cancer Res Clin Oncol 139(3): 367-378

Article

Oct 2012
J CANCER RES CLIN

Purpose: Although KRAS mutation has been identified as a negative predictive biomarker of anti-EGFR antibodies in metastatic colorectal cancer (mCRC), the efficacy in mCRC patients with KRAS wild-type status remains limited. Anti-EGFR antibodies work by blocking ligand binding, but the significance of EGFR ligands in mCRC has not been completely described. This study was conducted to identify the correlation between all seven EGFR ligands and clinical outcomes in mCRC treated with anti-EGFR antibodies. Furthermore, we determined an appropriate predictive strategy for anti-EGFR antibodies using these EGFR ligands. Methods: Among 36 mCRC patients who had been treated with cetuximab or panitumumab, we identified 26 mCRC patients with wild-type KRAS status treated properly as the second and further lines and analyzed the relationship between immunoreactivity to seven EGFR ligands and clinical outcomes. Results: Good clinical outcomes were associated with immunoreactivity against amphiregulin (AR), heparin-binding epidermal growth factor (HB-EGF), transforming growth factor-α (TGF-α), and epiregulin (EREG). Further, patients with immunoreactivity to greater than two of these four ligands (AR, HB-EGF, TGF-α, and EREG) had significantly higher response rate (53.3 vs. 0.0 %, p = 0.004) and disease control rate (93.3 vs. 9.0 %, p = 0.00002) and longer progression-free survival (median PFS: 231 vs. 79 days, p = 0.000008), when compared with patients with immunoreactivity against zero or one ligand. Conclusions: Immunohistochemical analysis of four EGFR ligands (AR, HB-EGF, TGF-α, and EREG) might be a novel predictive biomarker and may help optimize patient selection for cetuximab and panitumumab therapy in patients with mCRC.

Ectodomain shedding of TGF-α and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades

Article

Full-text available

Dec 1999
EMBO J

A variety of transmembrane proteins, such as transforming growth factor- (TGF-), tumor necrosis factor- (TNF-) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. We show that growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF- through activation of the Erk MAP kinase signaling cascade without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase was sufficient to stimulate TGF- shedding. The basal cleavage level in the absence of exogenous growth factor stimulation was due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activated TGF- shedding in the absence of exogenous stimuli. In addition to TGF- shedding, these mechanisms also mediate L-selectin and TNF- cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, was strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. Our results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins.

Chang D, Liu N, Yayon A, Wen D & Yarden Y. ErbB-3 and erbB-4 function as the respective low and high affinity receptors of all neu differentiation factor/heregulin isoforms

Article

Full-text available

Jan 1994

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4

Article

Full-text available

Jun 1998
P NATL ACAD SCI USA

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-β (NRG1-β), betacellulin (BTC), transforming growth factor-α (TGF-α), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-β and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-α was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-β or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-α and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcγRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.

Requirement for neuregulin receptor erbB2 in neural and cardiac development

Article

Full-text available

Nov 1995

THE receptor erbB2/neu is a member of the epidermal growth factor receptor (EGFR or erbB) family that also includes erbB3 and erbB41. Amplification of the erbB2/neu gene is found in many cancer types and its overexpression is correlated with a poor prognosis for breast and ovarian cancer patients2. Investigation of the biology of erbB2 led to the identification of a family of ligands termed neuregulins which included the neu-differentiation factors3,4, the heregulins5, a ligand with acetylcholine-receptor-inducing activity6 and glial growth factor7. Several lines of evidence suggest that heterodimerization of erbB2 with other erbB receptors is required for neuregulin signalling1. Here we investigate the developmental role of erbB2 in mammalian development in mice carrying an erbB2 null allele. We find that mutant embryos die before Ell, probably as a result of dysfunctions associated with a lack of cardiac trabeculae. Development of cranial neural-crest-derived sensory ganglia was markedly affected. Dil retrograde tracing revealed that the development of motor nerves was also compromised. Our results demonstrate the importance of erbB2 in neural and cardiac development.

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors

Article

Full-text available

Sep 1997
EMBO J

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D.E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3–EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D.E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

A metalloprotease-disintegrin, MDC9/meltrin-??/ADAM9 and PKC?? are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor

Article

Full-text available

Dec 1998
EMBO J

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKC binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-/ADAM9, a member of the metalloprotease–disintegrin family. Furthermore, the presence of constitutively active PKC or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKC, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKC are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation

Article

Full-text available

Sep 1975

Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.

The Primary Structure of Epidermal Growth Factor

Article

Full-text available

Jan 1973

The complete amino acid sequence of epidermal growth factor (EGF) has been established through the use of automated Edman degradation and standard enzymatic and chemical techniques. The location of the half-cystinyl residues was facilitated by the use of the S-[¹⁴C]aminoethyl derivative of EGF. The sequence is: [see PDF for sequence]. The calculated molecular weight of the 53-residue polypeptide is 6045, a value that is in agreement with the molecular weight of 6400 established by physical measurements. The peptide is acidic and the 6 half-cystines exist in disulfide linkage. Four arginine residues are located in the COOH-terminal portion of the molecule. The six COOH-terminal amino acids are not necessary for biological activity and EGF1–47 has activity identical with that of the 53-residue native molecule.

Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor

Article

Full-text available

Dec 1995

Various in vitro studies have suggested that ErbB4 (HER4) is a receptor for the neuregulins, a family of closely related proteins implicated as regulators of neural and muscle development, and of the differentiation and oncogenic transformation of mammary epithelia. Here we demonstrate that ErbB4 is an essential in vivo regulator of both cardiac muscle differentiation and axon guidance in the central nervous system (CNS). Mice lacking ErbB4 die during mid-embryogenesis from the aborted development of myocardial trabeculae in the heart ventricle. They also display striking alterations in innervation of the hindbrain in the CNS that are consistent with the restricted expression of the ErbB4 gene in rhombomeres 3 and 5. Similarities in the cardiac phenotype of ErbB4 and neuregulin gene mutants suggest that ErbB4 functions as a neuregulin receptor in the heart; however, differences in the hindbrain phenotypes of these mutants are consistent with the action of a new ErbB4 ligand in the CNS.

Coexpression of erbB2 and er6B3 proteins reconstitutes a high affinity receptor for heregulin

Article

Full-text available

Jun 1994

The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.

Autocrine transforming growth factor α is dispensible for v-rasHa-induced epidermal neoplasia: Potential involvement of alternate epidermal growth factor receptor ligands

Article

Full-text available

Jun 1995

Autocrine epidermal growth factor receptor activation by transforming growth factor alpha (TGF alpha) has been implicated in growth stimulation during epithelial neoplasia. Using keratinocytes isolated from mice with genetic defects in TGF alpha expression, we tested whether TGF alpha is required for transformation by the v-rasHa oncogene. Introduction of v-rasHa into primary epidermal cultures using a retroviral vector stimulated growth of both control (TGF alpha +/+, BALB/c) and TGF alpha-deficient (TGF alpha -/-, wa-1) keratinocytes. Moreover, v-rasHa elicited characteristic changes in marker expression (keratin 1 was suppressed; keratin 8 was induced), previously shown to be associated with epidermal growth factor (EGF) receptor activation, in both TGF alpha +/+ and TGF alpha -/- keratinocytes. v-rasHa markedly increased secreted (> 10-fold) and cell-associated (2-3-fold) TGF alpha levels in keratinocytes from TGF alpha +/+ and BALB/c mice, but not TGF alpha -/- or wa-1 mice. Based on Northern blot analysis, v-rasHa induced striking up-regulation of transcripts encoding the additional EGF family members amphiregulin, heparin-binding EGF-like growth factor, and betacellulin in cultured keratinocytes from all four mouse strains. Interestingly, in addition to the normal 4.5-kilobase TGF alpha transcript, wa-1 keratinocytes expressed two additional TGF alpha transcripts, 4.7 and 5.2 kilobases long. All three transcripts were up-regulated in response to v-rasHa, as well as exogenous TGF alpha or keratinocyte growth factor treatment, and were also detected in RNA isolated from wa-1 brain and skin. In vivo, v-rasHa keratinocytes from control as well as TGF alpha-deficient mice produced squamous tumors when grafted onto nude mice, and these lesions expressed high levels of amphiregulin, heparin-binding EGF-like growth factor, and betacellulin mRNA, regardless of their TGF alpha status. These findings indicate that TGF alpha is not essential for epidermal neoplasia induced by the v-rasHa oncogene and suggest that another EGF family member(s) may contribute to autocrine growth stimulation of ras-transformed keratinocytes.

Receptor Protein-Tyrosine Kinases and Their Signal Transduction Pathways

Article

Full-text available

Feb 1994
Annu Rev Cell Biol

ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all NDF/Heregulin isoforms

Article

Full-text available

Nov 1994

Neu differentiation factor (NDF or heregulin) elevates tyrosine phosphorylation of the ErbB-2 receptor tyrosine kinase, and it was, therefore, thought to function as a ligand of this receptor. However, several lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule, which belongs to the family of epidermal growth factor receptors. To address this question we constructed soluble chimeric proteins between alkaline phosphatase and the extracellular domains of ErbB-2 and either ErbB-3 or ErbB-4, two newly recognized members of the epidermal growth factor receptor family. Using the soluble proteins we found that beta isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors but not to the soluble ErbB-2 protein. When ectopically expressed in monkey fibroblasts, the full-length ErbB-3 and ErbB-4 receptors conferred specific binding to NDF. In these cells ErbB-3 displayed lower ligand binding affinity than ErbB-4, but like the latter receptor it preferred to bind the beta isoform over the alpha class of NDFs. These results indicate that both ErbB-3 and ErbB-4 function as physiological receptors of all NDF isoforms and suggest that a still unknown ligand of ErbB-2 exists.

Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family

Article

Full-text available

Oct 1994

Several polypeptide growth factors related to epidermal growth factor (EGF) have been identified recently, including transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), and betacellulin (BTC). These peptides all bind to the EGF receptor (EGFr). In an effort to understand redundancy within this peptide family and interactions among these related peptides, we compared the biological activities of EGF, TGF-alpha, AR, and HB-EGF in an EGF-responsive, nontransformed intestinal epithelial line (RIE-1) and also determined the effect of individual EGF-related peptides on the expression of related family members in these cells. TGF-alpha, AR, HB-EGF, and EGF were equipotent in stimulating [3H]thymidine incorporation by RIE-1 cells and bound the EGFr with equivalent affinity. Each EGF-related peptide induced the mRNA expression of the remaining family members, including BTC. HB-EGF and AR mRNAs were induced rapidly (within 30 min) and to a greater extent than TGF-alpha and BTC mRNAs, suggesting heterogeneity in the molecular mechanisms for induction. This same pattern was observed for all EGF-related peptides tested. A similar pattern of mRNA induction was observed in secondary cultures of human keratinocytes and in LIM1215 colon adenocarcinoma cells. Nuclear run-on analysis showed that induction of AR and HB-EGF is, at least in part, regulated at the level of gene transcription. Concurrent treatment with HB-EGF and cycloheximide resulted in superinduction of HB-EGF and AR, suggesting that these peptides are immediate early genes in RIE-1 cells. Our results demonstrate an equivalent biological response to EGF-related peptides in RIE-1 cells and further indicate that extensive auto-induction and cross-induction occur within the EGF-related peptide family in several EGF-responsive epithelial cell types.

Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships

Article

Full-text available

Apr 1993

The Neu/HER-2 receptor tyrosine kinase is overexpressed in some types of human adenocarcinomas, including tumors of the breast and the ovary. A 44 kDa glycoprotein that elevates tyrosine phosphorylation of Neu has been isolated and named Neu differentiation factor (NDF), or heregulin. Here we show that NDF affects tyrosine phosphorylation of Neu in human tumor cells of breast, colon and neuronal origin, but not in ovarian cells that overexpress the receptor. By using monoclonal antibodies (mAbs) to Neu, we found that the ovarian receptor is immunologically and biochemically similar to the mammary p185neu. Nevertheless, unlike breast-derived Neu, the ovarian protein did not display covalent cross-linking to radiolabeled NDF, and was devoid of ligand-induced association with phosphatidylinositol 3'-kinase. Direct binding analysis showed that NDF binds with high affinity (Kd approximately 10(-9) M) to mammary cells, but its weak association with ovarian cells is probably mediated by heparin-like molecules. Similar to the endogenous receptor, the ectopically overexpressed Neu of mammary cells, but not of ovarian and fibroblastic cells, exhibited elevated levels of NDF-induced phosphorylation and covalent cross-linking of the radiolabeled factor. Taken together, our results imply that NDF binding to cells requires both Neu and an additional cellular component, whose identity is still unknown, but its tissue distribution is more restricted than the expression of the neu gene.

Recombinant human betacellulin: Molecular structure, biological activities, and receptor interaction

Article

Full-text available

May 1994

Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR.

Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development

Article

Jun 1999

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFalpha-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFalpha in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.

An Essential Role for Ectodomain Shedding in Mammalian Development

Article

Nov 1998

Jacques J. Peschon

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-α converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-α (TNFα) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the l-selectin adhesion molecule, and transforming growth factor-α (TGFα). The phenotype of mice lacking TACE suggests an essential role for soluble TGFα in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Biological Response to ErbB Ligands in Nontransformed Cell Lines Correlates with a Specific Pattern of Receptor Expression

Article

Dec 1998

S. Sundaresan

The human epidermal growth factor receptor (HER or ErbB) family consists of four distinct members, including the epidermal growth factor (EGF) receptor (EGFR, HER1, or ErbB1), ErbB2 (HER2 or neu), ErbB3 (HER3), and ErbB4 (HER4). Activation of these receptors plays an important role in the regulation of cell proliferation, differentiation, and survival in several different tissues. Binding of a specific ligand to one of the ErbB receptors triggers the formation of specific receptor homo- and heterodimers, with ErbB2 being the preferred signaling partner. We analyzed the levels of various ErbB receptor messenger RNAs in a series of nontransformed cell lines by real time quantitative RT-PCR. The cell lines chosen were derived from a variety of tissues, including pancreas, lung, heart, and nervous system. Further, we measured biological responses in these cell lines upon treatment with EGF, betacellulin, and two types of neuregulins, heregulin and sensory and motor neuron-derived factor. All cell lines examined expressed detectable levels of ErbB2. High levels of expression of ErbB3 were correlated with responsiveness to heregulin and sensory and motor neuron-derived factor, whereas high levels of EGFR expression were correlated with responsiveness to EGF and betacellulin. Moreover, the sensitivity of a cell line to ErbB ligands was also correlated with the levels of expression of the appropriate ErbB receptors in that cell line. These results are consistent with our hypothesis that appropriate biological responsiveness to ErbB ligands is determined by the levels of expression of specific ErbB receptor combinations within a given tissue.

Receptor Protein-Tyrosine Kinases and Their Signal Transduction Pathways

Article

Jan 1994

Peter van der Geer

Tissue-Specific Knockout of the Insulin Receptor in Pancreatic ?? Cells Creates an Insulin Secretory Defect Similar to that in Type 2 Diabetes

Article

Feb 1999
CELL

Dysfunction of the pancreatic β cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the β cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the β cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic β cell and suggest that defects in insulin signaling at the level of the β cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.

Growth Factor-Mediated Proliferation and Differentiation of Insulin-Producing INS1 and RINm5F Cells: Identification of Betacellulin as a Novel Cell Mitogen

Article

Apr 1998
ENDOCRINOLOGY

It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic b-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epi- dermal growth factor (EGF) family that has not previously been shown to be mitogenic for b-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-b, platelet-derived growth factors, basic fibro- blast growth factor, EGF, transforming growth factor-a (TGF-a), neu differentiation factor, and TGF-b were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain dif- ferentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an in- hibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the b-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-a were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of b-cell growth and differen- tiation because the responses to previously identified b-cell mitogens were essentially similar to those reported in primary cells. In addi- tion, we have identified betacellulin as a possible modulator of b-cell growth. (Endocrinology 139: 1494 -1499, 1998)

Neuregulin-3 (NRG3): A novel neural tissue-enriched protein that binds and activates ErbB4

Article

Sep 1997
P NATL ACAD SCI USA

We describe the identification of Neuregulin-3 (NRG3), a novel protein that is structurally related to the neuregulins (NRG1). The NRG1/neuregulins are a diverse family of proteins that arise by alternative splicing from a single gene. These proteins play an important role in controlling the growth and differentiation of glial, epithelial, and muscle cells. The biological effects of NRG1 are mediated by receptor tyrosine kinases ErbB2, ErbB3, and ErbB4. However, genetic studies have suggested that the activity of ErbB4 may also be regulated in the central nervous system by a ligand distinct from NRG1. NRG3 is predicted to contain an extracellular domain with an epidermal growth factor (EGF) motif, a transmembrane domain, and a large cytoplasmic domain. We show that the EGF-like domain of NRG3 binds to the extracellular domain of ErbB4 in vitro. Moreover, NRG3 binds to ErbB4 expressed on cells and stimulates tyrosine phosphorylation of this receptor. The expression of NRG3 is highly restricted to the developing and adult nervous system. These data suggest that NRG3 is a novel, neural-enriched ligand for ErbB4.

Processing and juxtacrine activity of membrane‐anchored betacellulin

Article

Mar 1999
J CELL BIOCHEM

Betacellulin (BTC) was originally isolated as a secreted growth factor from a mouse pancreatic β-tumor cell line, whereas the cDNA sequence predicts that BTC is synthesized as a larger transmembrane protein. In the present study, we have characterized the membrane-anchored forms of BTC, using Chinese hamster ovary (CHO) cells, mouse fibroblast A9 cells, and a human breast cancer cell line MCF-7, all of which were stably transfected with human BTC cDNA. A9 and MCF-7 transfectants produced membrane-anchored BTC isoforms of 21, 25, 29, and 40 kDa on the cell surface, as well as a secreted BTC isoform. CHO transfectants secreted little BTC but accumulated the membrane-anchored isoforms. The cleavage of the membrane-anchored forms to release a secreted from of BTC was not enhanced by biological mediators such as a phorbol ester, which stimulates the cleavage of other membrane-anchored growth factors. The membrane-anchored forms of BTC expressed on the transfected cells induced the insulin production and/or promoted the growth in subclones of AR42J rat pancreatic cells. These results suggest that the membrane-anchored BTC can function as a juxtacrine factor in regulating the growth and differentiation of pancreatic endocrine cells. J. Cell. Biochem. 72:423–434, 1999. © 1999 Wiley-Liss, Inc.

Why are mammalian alkaline phosphatase much more active than bacterial alkaline phosphatases?

Article

May 1994
MOL MICROBIOL

Mammalian alkaline phosphatases are 20-30-fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active-site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site-specific mutagenesis, genetic and X-ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.

Regulation of signal diversity by receptor oligomerization

Article

Dec 1994
TRENDS BIOCHEM SCI

Receptor oligomerization was initially proposed as a mechanism by which epidermal growth factor activates the protein tyrosine kinase activity of its receptor. It is now well established that ligand-induced receptor oligomerization plays an important role in transmembrane signaling by a large number of receptors for hormones, cytokines and growth factors. Heterodimerization of the extracellular domains of two members of the same receptor family, or interaction with an accessory molecule, can increase the diversity of ligands recognized by individual receptors. Heterodimerization of cytoplasmic domains permits the recruitment of different complements of SH2-domain-containing signaling molecules, increasing the repertoire of signaling pathways that can be activated by a given receptor.

Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells

Article

Apr 1998

It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic beta-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epidermal growth factor (EGF) family that has not previously been shown to be mitogenic for beta-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-beta, platelet-derived growth factors, basic fibroblast growth factor, EGF, transforming growth factor-alpha (TGF-alpha), neu differentiation factor, and TGF-beta were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain differentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an inhibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the beta-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-alpha were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of beta-cell growth and differentiation because the responses to previously identified beta-cell mitogens were essentially similar to those reported in primary cells. In addition, we have identified betacellulin as a possible modulator of beta-cell growth.

Betacellulin, a member of the epidermal growth factor family, is overexpressed in human pancreatic cancer

Article

Oct 1995

Human pancreatic ductal adenocarcinomas overexpress the epidermal growth factor (EGF) receptor. Betacellulin is a mitogenic polypeptide that binds and activates this receptor. To determine whether betacellulin has a role in human pancreatic cancer, we studied its expression in cultured human pancreatic cancer cell lines and in normal and cancerous pancreatic tissues. Five of 6 pancreatic cancer cell lines expressed the 3 kb betacellulin mRNA moiety, T3M4, MiaPaCa-2 and COLO-357 cells exhibiting the highest expression levels. EGF, heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) increased betacelullin mRNA levels. Only 2 of 15 normal samples and 1 of 10 cancer samples failed to exhibit the betacellulin transcript. Densitometric analysis of the autoradiographs revealed a 7.5-fold increase in betacellulin mRNA levels in the cancer tissues by comparison with the normal tissues. By in situ hybridization, the duct-like cancer cells exhibited many betacellulin mRNA in situ hybridization grains. These findings indicate that human pancreatic cancer cells express betacellulin in culture and in vivo, and suggest that this EGF-like ligand may participate in aberrant autocrine and paracrine activation of the EGF receptor in human pancreatic cancer.

Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system

Article

Mar 1993

Glial growth factors, proteins that are mitogenic for Schwann cells, and several ligands for the p+/-85erbB2 receptor, are products of the same gene. Alternative splicing of the messenger RNA generates an array of putative membrane-attached, intracellular and secreted signalling proteins, at least some of which are expressed in the developing spinal cord and brain. These factors are probably important in the development and regeneration of the nervous system.

The mechanism of hydrolysis of ?? glycerophosphate by kidney alkaline phosphatase

Article

Oct 1975

Jan Ahlers

1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector.

Structure and Mechanism of Alkaline Phosphatase

Article

Feb 1992
ANNU REV BIOPH BIOM

J E Coleman

Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 Å (Zn1-Zn2), 4.9 Å (Zn2-Mg3), and 7.1 Å (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51 bridges Zn2 and Mg3. A crystal structure at 2.0-Å resolution of the noncovalent phosphate complex, E · P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E · P structure to the enzyme-substrate complex, E · ROPO42-, leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E · P intermediate is the slowest, 35 s-1 and therefore the rate-limiting, step of the mechanism at alkaline pH.

A heparin-binding EGF-like growth factor secreted by macrophage-like cells is related to EGF

Article

Mar 1991

Macrophage-like U-937 cells secrete a 22-kilodalton heparin-binding growth factor that is mitogenic for BALB-3T3 fibroblasts and smooth muscle cells, but not endothelial cells. The amino acid sequence predicted from complementary DNA clones indicates that the mitogen is a new member of the epidermal growth factor (EGF) family. This heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors on A-431 epidermoid carcinoma cells and smooth muscle cells, but is a far more potent mitogen for smooth muscle cells than is EGF. HB-EGF is also expressed in cultured human macrophages and may be involved in macrophage-mediated cellular proliferation.

Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis

Article

Apr 1991

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.

Signal Transduction by Receptors With Tyrosine Kinase Activity

Article

May 1990
CELL

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Structure and Function of Human Amphiregulin: A Member of the Epidermal Growth Factor Family

Article

Mar 1989

The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.

Mutationally altered rate constants in the mechanism of alkaline phosphatase

Article

Oct 1974

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.

Rat Transforming Growth Factor Type 1: Structure and Relation to Epidermal Growth Factor

Article

Apr 1984

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.

Mutations at Positions 153 and 328 inEscherichia coliAlkaline Phosphatase Provide Insight Towards the Structure and Function of Mammalian and Yeast Alkaline Phosphatases

Article

Dec 1995

In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.

Kavanaugh, W.M. & Williams, L.T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science 266, 1862-1865

Article

Jan 1995

Src homology 2 (SH2) domains bind specifically to tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes. A protein domain was identified that bound specifically to the tyrosine-phosphorylated form of its target protein but differs from known SH2 sequences. Phosphotyrosine-binding (PTB) domains were found in two proteins: SHC, a protein implicated in signaling through Ras; and SCK, encoded by a previously uncharacterized gene. The PTB domain of SHC specifically bound to a tyrosine-phosphorylated 145-kilodalton protein. PTB domains are an alternative to SH2 domains for specifically recruiting tyrosine-phosphorylated proteins into signaling complexes and are likely to take part in signaling by many growth factors.

Mouse Chromosomal Location of Three EGF Receptor Ligands: Amphiregulin (Areg), Betacellulin (Btc), and Heparin-Binding EGF (Hegfl)

Article

Aug 1995

The products of five distinct loci, Egf, Tgfa, Areg, Btc, and Hegfl act as ligands for the epidermal growth factor receptor. Egf and Tgfa have previously been mapped to mouse chromosomes 3 and 6, respectively, but the mouse chromosomal locations of Areg, Btc, and Hegfl have not been reported. Here, we show that Areg and Btc are tightly linked on mouse chromosome 5, while Hegfl maps to mouse chromosome 18. We also provide evidence that a putative sixth family member, Sdgf, is in fact a species variant of Areg. These results confirm and extend known relationships between mouse and human chromosomes, and they also provide new information regarding the evolution of this important gene family.

Strain-Dependent Epithelial Defects in Mice Lacking the EGF Receptor

Article

Aug 1995

Mice and cells lacking the epidermal growth factor receptor (EGFR) were generated to examine its physiological role in vivo. Mutant fetuses are retarded in growth and die at mid-gestation in a 129/Sv genetic background, whereas in a 129/Sv x C57BL/6 cross some survive until birth and even to postnatal day 20 in a 129/Sv x C57BL/6 x MF1 background. Death in utero probably results from a defect in the spongiotrophoblast layer of the placenta. Newborn mutant mice have open eyes, rudimentary whiskers, immature lungs, and defects in the epidermis, correlating with the expression pattern of the EGFR as monitored by beta-galactosidase activity. These defects are probably cell-autonomous because chimeric mice generated with EGFR-/- embryonic stem cells contribute small amounts of mutant cells to some organs. These results indicate that the EGFR regulates epithelial proliferation and differentiation and that the genetic background influences the resulting phenotype.

Targeted Disruption of Mouse EGF receptor: Effect of Genetic Background on Mutant Phenotype

Article

Aug 1995

Gene targeting was used to create a null allele at the epidermal growth factor receptor locus (Egfr). The phenotype was dependent on genetic background. EGFR deficiency on a CF-1 background resulted in peri-implantation death due to degeneration of the inner cell mass. On a 129/Sv background, homozygous mutants died at mid-gestation due to placental defects; on a CD-1 background, the mutants lived for up to 3 weeks and showed abnormalities in skin, kidney, brain, liver, and gastrointestinal tract. The multiple abnormalities associated with EGFR deficiency indicate that the receptor is involved in a wide range of cellular activities.

Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor

Article

Aug 1995

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.

Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes

Article

Apr 1995

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

Hynes NE, Stern DFThe biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1198: 165-184

Article

Jan 1995
Biochim Biophys Acta

Modular binding domains in signal transduction

Article

Feb 1995
CELL

The transduction of a signal is a change in form of the signal as it is passed from one carrier to another. The root "duce" means "to lead" in Latin; thus, a signal is led through a cell by steps of transduction (the same root is in the words seduce and duct as well as II Duce). The earliest transduction steps that were elucidated involved massive release of small molecule "second messengers", originally cAMP, that flooded a cell with information. With the understanding that such proteins as tyrosine kinases and Ras relatives are signal transducers, came the realization that many signaling pathways are more precise, sending controlled and probably weakly amplified signals to specific targets. These intracellular signals are often maintained in macromolecular form rather than being passed to small molecules.

Heregulin induces tyrosine phosphorylation of HER4/p180erbB4

Article

Jan 1994

The HER4/ERBB4 gene encodes a 180K transmembrane protein (HER4/p180erbB4) that is structurally related to the 185K product (HER2/p185erbB2) of the HER2/ERBB2 proto-oncogene. A 45K heparin-binding glycoprotein (p45) has been characterized that specifically activates the intrinsic tyrosine kinase activity of HER4 (ref. 2). This HER4 ligand shares several features with the heregulin family of proteins, including molecular mass, ability to induce differentiation of breast cancer cells, activation of tyrosine phosphorylation in MDA-MB453 cells, and amino-terminal protein sequence. Heregulin exists as multiple isoforms and all are presumed to interact directly with HER2 (refs 3-6). We have used binding and phosphorylation studies with recombinant ligand on cell lines expressing recombinant receptors, and report here that heregulin, like p45, is a specific ligand for HER4. Furthermore, heregulin fails to induce phosphorylation of HER2 in the absence of HER4. These findings suggest that activation of the HER4 receptor is involved in signal transduction by heregulin.

Conversion of a magnesium binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase

Article

Nov 1993

The replacement of aspartic acid by histidine at position 153 in Escherichia coli alkaline phosphatase results in a mutant enzyme that is remarkably similar to certain mammalian alkaline phosphatases that are activated by magnesium in a time-dependent fashion. These mammalian alkaline phosphatases have histidine at the position corresponding to 153 of the E. coli sequence. Here we report the three-dimensional structure of the mutant E. coli alkaline phosphatase with histidine at position 153. The structure reveals that the octahedral magnesium binding site has been converted to a tetrahedral zinc binding site with an imidazole ring nitrogen of His-153 as one of the ligands to the zinc. The alteration in metal binding caused by the mutation could explain the origin of the magnesium activation observed with the mammalian alkaline phosphatases. The structure also reveals differences in the mode of phosphate binding, explaining the enhanced phosphate affinity and the reduced activity of the mutant enzyme in the presence of zinc.

Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structure stabilization and catalysis

Article

Mar 1993

Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase. The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions. However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme. The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme. Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+. The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change. The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values. The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme. Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and Expression of cDNA Encoding Human Betacellulin, a New Member of the EGF Family

Article

Mar 1993

Betacellulin (BTC) is a novel growth factor purified from the conditioned media of mouse pancreatic beta tumor cells and has been found to be a new member of the epidermal growth factor (EGF) family. The cDNA encoding human BTC has been cloned from a cDNA library prepared from human breast adenocarcinoma cell line MCF-7. The nucleotide sequence encodes a polypeptide which consists of 178 amino acid residues including a putative signal sequence, indicating that the structural organization of human BTC is similar to that of mouse BTC. The amino acid sequence of the human BTC precursor protein exhibits 79% similarity with that of the mouse precursor protein. The BTC gene was found to be expressed in several mouse tissues including kidney and liver as well as in a mouse beta tumor cell line and MCF-7 cells, suggesting that BTC might play a physiological role in normal tissues.

Structure-function and biological role of betacellulin

Abstract

No full-text available

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