ERK5 regulates glucose-induced increased fibronectin production in the endothelial cells and in the retina in diabetes.
ABSTRACT PURPOSE: Fibronectin (FN) production and deposition in the tissue is a characteristic feature of diabetic retinopathy. ERK5 is a recent member of MAPK family which plays a critical role in cardiovascular development and maintaining endothelial cell integrity. The aim of the study is to investigate the role of ERK5 signaling in glucose-induced FN overproduction. METHOD: Dermal-derived human microvascular endothelial cells (HMVECs) and human retinal microvascular endothelial cells (HRMECs) were used in this study. FN mRNA levels and secreted FN protein levels were measured using real-time PCR and ELISA respectively. Constitutively active MEK5 (CAMEK5) adenovirus was used to upregulate ERK5. Dominant negative MEK5 (DNMEK5) and ERK5 siRNA (siERK5) were used to downregulate ERK5. In parallel retinal tissues of diabetic rats were examined. Results: A significant decrease of FN was observed at both protein and mRNA levels following CAMEK5 transduction in basal as well as in high glucose. Dominant negative MEK5 (DNMEK5) transduction led to further enhancement of glucose-induced increased FN expression. ERK5 siRNA (siERK5) treatment led to an increase of FN synthesis. Retinal tissues of diabetic rats showed FN upregulation and ERK5 downregulation. TGFβ1 mRNA and phosphorylated Smad2 were markedly suppressed by CAMEK5 transduction with and without glucose treatment. On the other hand siERK5 transfection enhanced TGFβ1 mRNA expression. Exogenous NGF supplementation resulted in elevated phosphorylated and total ERK5 with and without glucose treatment. CONCLUSION: our experiments demonstrated a novel mechanism of glucose-induced increased FN production which is mediated through decreased ERK5 signaling.
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ABSTRACT: Background: Podocytes are highly specialized cells integral to the normal functioning kidney, however, in diabetic nephropathy injury occurs leading to a compromised phenotype and podocyte dysfunction which critically produces podocyte loss with subsequent renal impairment. TGFβ1 holds a major role in the development of diabetic nephropathy. Erk5 is an atypical mitogen-activated protein (MAP) kinase involved in pathways modulating cell survival, proliferation, differentiation, and motility. Accordingly, the role of Erk5 in mediating TGFβ1-induced podocyte damage was investigated. Methods: Conditionally immortalized human podocytes were stimulated with TGFβ1 (2.5 ng/ml); inhibition of Erk5 activation was conducted with the chemical inhibitor BIX02188 (10 μM) directed to the upstream Mek5; inhibition of Alk5 was performed with SB431542 (10 μM); Ras signaling was inhibited with farnesylthiosalicylic acid (10 μM). Intracellular signaling proteins were investigated by western blotting; phenotype was explored by immunofluorescence; proliferation was assessed with a MTS assay; motility was examined with a scratch assay; barrier function was studied using electric cell-substrate impedance sensing; apoptosis was studied with annexin V-FITC flow cytometry. Results: Podocytes expressed Erk5 which was phosphorylated by TGFβ1 via Mek5, whilst not involving Ras. TGFβ1 altered podocyte phenotype by decreasing P-cadherin staining and increasing α-SMA, as well as reducing podocyte barrier function; both were prevented by inhibiting Erk5 phosphorylation with BIX02188. TGFβ1-induced podocyte proliferation was prevented by BIX02188, whereas the induced apoptosis was not. Podocyte motility was reduced by BIX02188 alone and further diminished with TGFβ1 co-incubation. Conclusion: These results describe for the first time the expression of Erk5 in podocytes and identify it as a potential target for the treatment of diabetic renal disease.Frontiers in Pharmacology 04/2014; 5:71.
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ABSTRACT: Lithospermic acid B (LAB), an active component isolated from Salvia miltiorrhiza radix, has been reported to have antioxidant effects. We examined the effects of LAB on the prevention of diabetic retinopathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes.PLoS ONE 06/2014; 9(6):e98232. · 3.53 Impact Factor
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ABSTRACT: Endothelial cell (EC) damage is a key mechanism causing retinal microvascular injury in diabetes. Several microRNAs (miRNAs) have been found to regulate sirtuin 1 (SIRT1, which is involved in regulation of the cell cycle, survival and metabolism) in various tissues and disease states, but no studies have been conducted on the role of miRNA in regulation of SIRT1 in diabetic retinopathy. Here we investigated the effect of miRNA-195 (miR-195), a SIRT1-targeting miRNA, on the development of diabetes-induced changes in ECs and retina. The level of miR-195 was measured in human retinal and dermal microvascular ECs (HRECs, HMECs) following exposure to 25 mmol/l glucose (high glucose, HG) and 5 mmol/l glucose (normal glucose, NG). SIRT1 and fibronectin levels were examined following transfection with miR-195 mimic or antagomir or forced expression of SIRT1. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of an miR-195 antagomir or mimic. In situ hybridisation was used to localise retinal miR-195. HG caused increased miR-195 levels and decreased SIRT1 expression (compared with NG) in both HRECs and HMECs. Transfection with miR-195 antagomir and forced expression of SIRT1 prevented such changes, whereas transfection with miR-195 mimic produced HG-like effects. A luciferase assay confirmed the binding of miR-195 to the 3' untranslated region of SIRT1. miR-195 expression was upregulated in retinas of diabetic rats and intravitreal injection of miR-195 antagomir ameliorated levels of SIRT1. These studies identified a novel mechanism whereby miR-195 regulates SIRT1-mediated tissue damage in diabetic retinopathy.Diabetologia 02/2014; · 6.88 Impact Factor