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ABSTRACT: Abstract Diabetes mellitus is commonly considered as a disease of a scant β-cell mass that fails to respond adequately to the functional demand. Tyrosine kinases may play a role for β-cell replication, differentiation (neoformation) and survival. Transfection of β-cells with DNA constructs coding for tyrosine kinase receptors yields a ligand-dependent increase of DNA synthesis in β-cells. A PCR-based technique was adopted to assess the repertoire of tyrosine kinases expressed in fetal islet-like structures, adult islets or RINm5F cells. Several tyrosine kinase receptors, such as the VEGFR-2 (vascular endothelial growth factor receptor 2) and c-Kit, were found to be present in pancreatic duct cells. Because ducts are thought to harbor β-cell precursor cells, these receptors may play a role for the neoformation of β-cells. The Src-like tyrosine kinase mouse Gtk (previously named Bsk/Iyk) is expressed in islet cells, and was found to inhibit cell proliferation. Furthermore, it conferred decreased viability in response to cytokine exposure. Shb is a Src homology 2 domain adaptor protein which participates in tyrosine kinase signaling. Transgenic mice overexpressing Shb in β-cells exhibit an increase in the neonatal β-cell mass, an improved glucose homeostasis, but also decreased survival in response to cytokines and streptozotocin. It is concluded that tyrosine kinase signaling may generate multiple responses in β-cells, involving proliferation, survival and differentiation.
03/2011; 105(2):7-15.
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ABSTRACT: Abstract Background Shb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and development. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function. Results Shb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ TH cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production of naïve TH cells. This suggests a TH2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern. Conclusion Our results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ TH2 cell response.
BMC Immunology. 01/2011;
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ABSTRACT: Shb, a ubiquitously expressed Src homology 2 domain-containing adaptor protein has previously been implicated in the signaling of various tyrosine kinase receptors including the TCR. Shb associates with SLP76, LAT and Vav, all important components in the signaling cascade governing T cell function and development. A Shb knockout mouse was recently generated and the aim of the current study was to address the importance of Shb deficiency on T cell development and function.
Shb knockout mice did not display any major changes in thymocyte development despite an aberrant TCR signaling pattern, including increased basal activation and reduced stimulation-induced phosphorylation. The loss of Shb expression did however affect peripheral CD4+ T(H) cells resulting in an increased proliferative response to TCR stimulation and an elevated IL-4 production of naïve T(H) cells. This suggests a T(H)2 skewing of the Shb knockout immune system, seemingly caused by an altered TCR signaling pattern.
Our results indicate that Shb appears to play an important modulating role on TCR signaling, thus regulating the peripheral CD4+ T(H)2 cell response.
BMC Immunology 01/2011; 12:3. · 2.53 Impact Factor
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ABSTRACT: Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2 (VEGFR-2). Previous experiments have suggested a role for Shb in endothelial cell function. Recently, the Shb gene was inactivated and Shb null mice were obtained on a mixed genetic background, but not on C57Bl6 mice. The present study was performed to address endothelial function in the Shb knockout mouse and its relevance for tumor angiogenesis. Tumor growth was retarded in Shb mutant mice, and this correlated with decreased angiogenesis both in tumors and in Matrigel plugs. Shb null mice display an abnormal endothelial ultrastructure in liver sinusoids and heart capillaries with cytoplasmic extensions projecting toward the lumen. Shb null heart VE-cadherin staining was less distinct than that of control heart, exhibiting in the former case a wavy and punctuate pattern. Experiments on isolated endothelial cells suggest that these changes could partly reflect cytoskeletal abnormalities. Vascular permeability was increased in Shb null mice in heart, kidney, and skin, whereas VEGF-stimulated vascular permeability was reduced in Shb null mice. It is concluded that Shb plays an important role in maintaining a functional vasculature in adult mice, and that interference with Shb signaling may provide novel means to regulate tumor angiogenesis.
Cancer Research 03/2009; 69(5):2141-8. · 7.86 Impact Factor
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ABSTRACT: SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.
Developmental Dynamics 10/2007; 236(9):2485-92. · 2.54 Impact Factor
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ABSTRACT: Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.
Journal of Biological Chemistry 12/2006; 281(45):34484-91. · 4.77 Impact Factor
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ABSTRACT: The potential use of embryonic stem (ES) cells for cell therapy of diabetes requires improved methods for differentiation and isolation of insulin-producing beta-cells. The signal transduction protein SHB may be involved in both angiogenesis and beta-cell development. Here we show that cells expressing the pancreatic endodermal marker PDX-1 appear in the vicinity of vascular structures in ES cell-derived embryoid bodies (EBs) cultured in vitro. Moreover, overexpression of SHB as well as culture of EBs in presence of the angiogenic growth factors PDGF or VEGF enhanced the expression of PDX-1 and/or insulin mRNA. Finally, expression of GFP under control of the PDX-1 promoter in EBs allowed for the enrichment by FACS of cells expressing PDX-1, C-peptide, and insulin as determined by immunofluorescence. It is concluded that SHB and angiogenic factors promote the development of cells expressing PDX-1 and insulin in EBs and that such cells can be separated by FACS.
Biochemical and Biophysical Research Communications 07/2006; 344(2):517-24. · 2.48 Impact Factor
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ABSTRACT: Embryonic stem (ES) cells represent an attractive tool not only for the study of the development of various cell types but also as a potential source of cells for transplantation. Previous studies suggested a role of the signal transduction protein SRC homology 2(SH2) protein of Beta-cells (SHB) for the development of both pancreatic 3-cells and blood vessels. SHB is an SH2 domain-containing adapter protein involved in the generation of signaling complexes in response to activation of a variety of receptors, several of which have been implicated in developmental processes. Moreover, microarray analysis of ES cells expressing mutant SHB has revealed decreased expression of several genes of developmental importance. Here, we present protocols that may be used for transfection of mouse ES cells and to study the differentiation of ES cell-derived embryoid bodies (EBs) into the pancreatic Beta-cell lineage as well as into vascular structures with special reference to the effect of SHB. Moreover, we also provide a protocol that may be used for enrichment by fluorescence-activated cell sorting of specific cell lineages in EBs.
Methods in molecular biology (Clifton, N.J.) 02/2006; 330:353-72.
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ABSTRACT: An important reason for the large amount of islets required for successful islet transplantation is likely to be inadequate engraftment of the transplanted islets. Thus, the revascularization is of major importance for graft survival. In order to study the expression of angiogenic peptides and receptors on islet endothelial cells (EC), we needed methods giving access to such endothelium. Therefore, we developed methods to isolate EC from islets transplanted intraportally or beneath the kidney capsule.
Pancreatic islets were isolated, cultured and syngeneically transplanted into the liver or beneath the kidney capsule of C57BL/6 mice. One month post-transplantation, the islets were retrieved and EC from these islets were explanted. EC were also collected from freshly isolated and cultured non-transplanted islets. The EC were purified with Dynabeads and identified with immunocytochemistry. Angiogenesis GEArray technology was used to study angiogenic gene expression.
Several angiogenic genes were expressed in EC; e.g. endostatin, pigment-epithelial derived factor, vascular endothelial growth factor and angiopoietin-2, and their expression were affected by culture.
The expression of angiogenesis-related genes in islet EC from non-transplanted islets is affected by culture. Moreover, we also describe a technique, which makes it possible to obtain EC from transplanted islets.
Pancreatology 02/2006; 6(1-2):86-95. · 1.99 Impact Factor
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ABSTRACT: Embryonic stem (ES) cells represent an attractive tool not only for the study of the development of various cell types but
also as a potential source of cells for transplantation. Previous studies suggested a role of the signal transduction protein
SRC homology 2(SH2) protein of Beta-cells (SHB) for the development of both pancreatic β-cells and blood vessels. SHB is an
SH2 domain-containing adapter protein involved in the generation of signaling complexes in response to activation of a variety
of receptors, several of which have been implicated in developmental processes. Moreover, microarray analysis of ES cells
expressing mutant SHB has revealed decreased expression of several genes of developmental importance. Here, we present protocols
that may be used for transfection of mouse ES cells and to study the differentiation of ES cell-derived embryoid bodies (EBs)
into the pancreatic β-cell lineage as well as into vascular structures with special reference to the effect of SHB. Moreover,
we also provide a protocol that may be used for enrichment by fluorescenceactivated cell sorting of specific cell lineages
in EBs.
Key Wordsβ-cell–CD31–development–ES cells–FACS–β-galactosidase–green fluorescent–protein–insulin–pancreas–PDX-1–SHB–vasculogenesis
12/2005: pages 353-372;
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ABSTRACT: Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.
Current Molecular Medicine 07/2003; 3(4):313-24. · 5.10 Impact Factor
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ABSTRACT: The SH2 domain-containing adapter protein SHB transmits signals from receptor tyrosine kinases regulating diverse processes such as apoptosis and differentiation. To elucidate a role for SHB in cell differentiation, wild-type and R522K (inactive SH2 domain-mutant) SHB were transfected and expressed in mouse embryonic stem (ES) cells. Microarray analysis using Affymetrix U74A chips on undifferentiated ES cells and expression of selected differentiation markers after generation of embryoid bodies were subsequently assessed. Wild-type SHB altered the expression of 16 genes in undifferentiated ES cells, many of which have been found to relate to neural cell function. R522K-SHB altered the expression of 128 genes in undifferentiated ES cells, the majority of which were decreased, including several transcription factors related to development. When grown as embryoid bodies, after 4 days R522K-SHB ES cells were already found to display a different morphological appearance, with an impaired cavity formation that occurred in the absence of altered OCT4 expression. This impairment was reversed by exogenous addition of Matrigel. In addition, R522K-SHB embryoid bodies displayed reduced mRNA contents of the liver protein albumin, the pancreatic proteins amylase, glucagon and insulin after 20 days of differentiation. Matrigel did not restore the impaired expression of albumin in the R522K-SHB cells. Expression of the mesodermal marker cardiac actin and the neural marker neurofilament heavy chain alpha was not affected by wild-type or R522K-SHB overexpression. It is concluded that SHB is required for efficient differentiation of ES cells into embryoid bodies with normal cavities and cells belonging to endodermal lineages.
Experimental Cell Research 06/2003; 286(1):40-56. · 3.58 Impact Factor
