MicroRNA-125b (miR-125b) has been implicated in a variety of diseases as either repressors or promoters, and plays crucial roles in many cellular processes such as cell differentiation, proliferation and apoptosis. Age-related cataract has become one of the most serious problems facing the aging population in the world. The purpose of this study was to investigate the role of miR-125b in the development of age-related cataract. We demonstrated that miR-125b was downregulated in both age-related cataract tissue and lens epithelial cell apoptosis induced by UV irradiation. We also identified the impact of miR-125b on apoptosis in a lens epithelial cell line. In vitro, miR-125b regulates human lens epithelial cell apoptosis at least in part by directly targeting p53. In addition, an inverse relationship between miR-125b and p53 expression was seen in age-related cataract tissue. In conclusion, this study suggests that miR-125b might be closely involved in the pathogenesis of cataract, and has the potential to be a diagnostic biomarker or even a therapeutic modality for cataract.
Dupuytren's disease (DD) is a common fibrotic condition of the palmar fascia, leading to deposition of collagen-rich cords and progressive flexion of the fingers. The molecular mechanisms underlying the disease are poorly understood. We have previously shown altered expression of extracellular matrix-degrading proteases (matrix metalloproteases, MMPs, and 'a disintegrin and metalloprotease domain with thrombospondin motifs', ADAMTS, proteases) in palmar fascia from DD patients compared to control and shown that the expression of a sub-set of these genes correlates with post-operative outcome. In the current study we used an in vitro model of collagen contraction to identify the specific proteases which mediate this effect. We measured the expression of all MMPs, ADAMTSs and their inhibitors in fibroblasts derived from the palmar fascia of DD patients, both in monolayer culture and in the fibroblast-populated collagen lattice (FPCL) model of cell-mediated contraction. Key proteases, previously identified in our tissue studies, were expressed in vitro and regulated by tension in the FPCL, including MMP1, 2, 3, 13 and 14. Knockdown of MMP2 and MMP14 (but not MMP1, 3 and 13) inhibited cell-mediated contraction, and knockdown of MMP14 inhibited proMMP-2 activation. Interestingly, whilst collagen is degraded during the FPCL assay, this is not altered upon knockdown of any of the proteases examined. We conclude that MMP-14 (via its ability to activate proMMP-2) and MMP-2 are key proteases in collagen contraction mediated by fibroblasts in DD patients. These proteases may be drug targets or act as biomarkers for disease progression.
Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation.
Sirt3, a mitochondrial NAD(+)-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F(o)F(1)ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F(o)F(1)ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F(o)F(1)ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F(o)F(1)ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F(o)F(1)ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ≅85% of mtDNA with 4977bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F(o)F(1)ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5-10μM of menadione impaired the Sirt3-mediated deacetylation and activation on F(o)F(1)ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F(o)F(1)ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.
Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remains to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation was also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.
Osteoporosis is a major public health issue that is expected to rise as the global population ages. Resveratrol (RES) is a plant polyphenol with various anti-aging properties. RES treatment of bone cells results in protective effects, but dose translation from in vitro studies to clinically relevant doses is limited since bioavailability is not taken into account. The aims of this review is to evaluate in vivo evidence for a role of RES supplementation in promoting bone health to reduced osteoporosis risk and potential mechanisms of action. Due to multiple actions on both osteoblasts and osteoclasts, RES has potential to attenuate bone loss resulting from different etiologies and pathologies. Several animal models have investigated the bone protective effects of RES supplementation. Ovariectomized rodent models of rapid bone loss due to estrogen-deficiency reported that RES supplementation improved bone mass and trabecular bone without stimulating other estrogen-sensitive tissues. RES supplementation prior to age-related bone loss was beneficial. The hindlimb unloaded rat model used to investigate bone loss due to mechanical unloading showed RES supplementation attenuated bone loss in old rats, but had inconsistent bone effects in mature rats. In growing rodents, RES increased longitudinal bone growth, but had no other effects on bone. In the absence of human clinical trials, evidence for a role of RES on bone heath relies on evidence generated by animal studies. A better understanding of efficacy, safety, and molecular mechanisms of RES on bone will contribute to the determination of dietary recommendations and therapies to reduce osteoporosis. This article is part of a Special Issue entitled: Resveratol: Challenges in translating pre-clincial findigns to iproved patient outcomes.
Emerging evidences suggest that chronic inflammation is one of the major causes of tumorigenesis. The role of inflammation in regulation of breast cancer progression is not well established. Recently Mediator of IRF3 Activation (MITA) protein has been identified that regulates NF-κB and IFN pathways. Role of MITA in the context of inflammation and cancer progression has not been investigated. In the current report, we studied the role of MITA in the regulation of cross talk between cell death and inflammation in breast cancer cells. The expression of MITA was significantly lower on in estrogen receptor (ER) positive breast cancer cells than ER negative cells. Similarly, it was significantly down regulated in tumor tissue as compared to the normal tissue. The overexpression of MITA in MCF-7 and T47D decreases the cell proliferation and increases the cell death by activation of caspases. MITA positively regulates NF-κB transcription factor, which is essential for MITA induced cell death. The activation of NF-κB induces TNF-α production which further sensitizes MITA induced cell death by activation of death receptor pathway through capsase-8. MITA expression decreases the colony forming units and migration ability of MCF-7 cells. Thus, our finding suggests that MITA act as a tumor suppressor which is down regulated during tumorigenesis providing survival advantage to tumor cell.
We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.
Adiponectin (APN), an adipokine, exerts an anti-inflammatory and anti-cancerous activity with its role in glucose and lipid metabolism and its absence related to several obesity related malignancies including colorectal cancer. The aim of this study is to determine the effect of APN deficiency on the chronic inflammation-induced colon cancer. This was achieved by inducing inflammation and colon cancer in both APN knockout (KO) and C57B1/6 wild type (WT) mice. They were divided into four treatment groups (n=6): 1) control (no treatment); 2) treatment with three cycles of dextran sodium sulfate (DSS); 3) weekly doses of 1,2-dimethylhydrazine (DMH) (20mg/kg of mouse body weight) for twelve weeks; 4) a single dose of DMH followed by 3 cycles of DSS (DMH+DSS). Mice were observed for diarrhea, stool hemoccult, and weight loss and were sacrificed on day 153. Tumor area and number were counted. Colonic tissues were collected for Western blot and immunohistochemistry analyses. APNKO mice were more protected than WT mice from DSS induced colitis during first DSS cycle, but lost this protection during the second and the third DSS cycles. APNKO mice had significantly severe symptoms and showed greater number and larger area of tumors with higher immune cell infiltration and inflammation than WT mice. This result was further confirmed by proteomic study including pSTAT3, pAMPK and Cox-2 by western blot and Immunohistochemistry. Conclusively, APN deficiency contributes to inflammation-induced colon cancer. Hence, APN may play an important role in colorectal cancer prevention by modulating genes involved in chronic inflammation and tumorigenesis.
Rapid remodeling of the actin cytoskeleton in the pre- and/or post-synaptic compartments is responsible for the regulation of neuronal plasticity, which is an important process for learning and memory. Cofilin1 plays an essential role in these processes and a dysregulation of its activity was associated with the cognitive decline observed during normal ageing and Alzheimer's disease (AD). To understand the mechanism(s) regulating Cofilin1 activity we evaluated changes occurring with regard to Cofilin1 and its up-stream regulators Lim kinase-1 (LIMK1) and Slingshot phosphatase-1 (SSH1) in (i) human AD brain, (ii) 1-, 4-, and 10-months old APP/PS1 mice, (iii) wild type 3-, 8-, 12-, 18- and 26-months old mice, as well as in cellular models including (iv) mouse primary cortical neurons (PCNs, cultured for 5, 10, 15 and 20days in vitro) and (v) mouse embryonic fibroblasts (MEF). Interestingly, we found an increased Cofilin1 phosphorylation/inactivation with age and AD pathology, both in vivo and in vitro. These changes were associated with a major inactivation of SSH1. Interestingly, inhibition of γ-secretase activity with Compound-E (10μM) prevented Cofilin1 phosphorylation/inactivation through an increase of SSH1 activity in PCNs. Similarly, MEF cells double knock-out for γ-secretase catalytic subunits presenilin-1 and -2 (MEFDKO) showed a strong decrease of both Cofilin1 and SHH1 phosphorylation, which were rescued by the overexpression of human γ-secretase. Together, these results shed new light in understanding the molecular mechanisms promoting Cofilin1 dysregulation, both during ageing and AD. They further have the potential to impact the development of therapies to safely treat AD.
Alzheimer's disease (AD) is a devastating disorder that strikes 1 in 10 Americans over the age of 65, and almost half of all Americans over 85years old. The odds of an individual developing Alzheimer's disease double every five years after the age of 65. While it has become increasingly common to meet heart attack or cancer survivors, there are no Alzheimer's disease survivors. There is mounting evidence that dietary polyphenols, including resveratrol, may beneficially influence Alzheimer's disease (AD). Based on this consideration, several studies reported in the last few years were designed to validate sensitive and reliable translational tools to mechanistically characterize brain bioavailable polyphenols as disease-modifying agents to help prevent the onset of AD dementia and other neurodegenerative disorders. Several research groups worldwide with expertise in AD, plant biology, nutritional sciences, and botanical sciences have reported very high quality studies that ultimately provided the necessary information showing that polyphenols and their metabolites, which come from several dietary sources, including grapes, cocoa etc., are capable of preventing AD. The ultimate goal of these studies was to provide novel strategies to prevent the disease even before the onset of clinical symptoms. The studies discussed in this review article provide support that the information gathered in the last few years of research will have a major impact on AD prevention by providing vital knowledge on the protective roles of polyphenols, including resveratrol.
Vascular endothelial cells (ECs) are responsible for post-ischemic angiogenesis, a process that is regulated by reactive oxygen species. Recent studies indicate that endothelial Nox4 based NADPH oxidase may have a key role. This study examines the role of endothelial Nox4 in ischemia-induced angiogenesis and explores the potential mechanisms involved. Mouse lines overexpressing human Nox4 wild type (EWT) or its dominant negative form P437H (EDN) specifically in the endothelium were used. Non-transgenic littermate mice (NTg) were used as controls. Following hind limb ischemia, blood flow recovery was enhanced in EWT and was impaired in EDN compared with NTg. The critical angiogenesis regulating genes vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS) and transforming growth factor β1 (TGFβ1) were upregulated in EWT both in the ischemic muscle and in heart ECs, while TGFβ1 was downregulated in EDN ECs. In EC, both VEGFA and TGFβ1 stimulated EC proliferation, migration, and capillary-like network formation in EWT but failed to do so in EDN. Application of TGFβ1 increased both VEGFR2 and eNOS expression levels, whereas blocking TGFβ1 or addition of catalase inhibited the phosphorylation of VEGFR2 and eNOS, indicating H2O2 and TGFβ1 signaling downstream of Nox4 is critical to maintain EC angiogenic functions. Use of cell specific transgenic mice with both upregulation and downregulation of endothelial Nox4 indicate several mechanisms linked to Nox4 play a role in angiogenesis. Endothelial Nox4 regulates ischemia-induced angiogenesis, likely through H2O2- and TGFβ1-mediated activation of cell signaling pathways essential for endothelial function.