ERK5 Regulates Glucose-Induced Increased Fibronectin Production in the Endothelial Cells and in the Retina in Diabetes

Department of Pathology, University of Western Ontario, London, ON, Canada.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 11/2012; 53(13). DOI: 10.1167/iovs.12-10553
Source: PubMed

Fibronectin (FN) production and deposition in the tissue is a characteristic feature of diabetic retinopathy. ERK5 is a recent member of the mitogen activated protein kinase (MAPK) family, which plays a critical role in cardiovascular development and maintaining endothelial cell integrity. The aim of this study was to investigate the role of ERK5 signaling in glucose-induced FN overproduction.

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 MAPK/ERK kinase 5 (MEK5 [CAMEK5]) adenovirus was used to upregulate ERK5. Dominant negative MEK5 (DNMEK5) and ERK5 siRNA (siERK5) were used to downregulate ERK5. Parallel retinal tissues of diabetic rats were examined.

A significant decrease of FN was observed at both protein and mRNA levels following CAMEK5 transduction in basal as well as in high glucose. DNMEK5 transduction led to further enhancement of glucose-induced increased FN expression. 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 nerve growth factor supplementation resulted in elevated phosphorylated and total ERK5 with and without glucose treatment.

Our experiments demonstrated a novel mechanism of glucose-induced increased FN production in diabetic retinopathy, which is mediated through decreased ERK5 signaling.

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    • "LAB treatment prevented diabetic nephropathy by inhibiting reactive oxygen species generation, protein kinase C (PKC) activation, and transforming growth factor (TGF)-β1 and fibronectin expression [22]. Considering similar phenomena are involved in the pathogenesis of diabetic retinopathy [25], [26], LAB treatment might serve as a potential preventive therapeutic measure for diabetic retinopathy. "
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    ABSTRACT: Background 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. Methods and Findings LAB (10 or 20 mg/kg) or normal saline were given orally once daily to 24-week-old male OLETF rats for 52 weeks. At the end of treatment, fundoscopic findings, vascular endothelial growth factor (VEGF) expression in the eyeball, VEGF levels in the ocular fluid, and any structural abnormalities in the retina were assessed. Glucose metabolism, serum levels of high-sensitivity C-reactive protein (hsCRP), monocyte chemotactic protein-1 (MCP1), and tumor necrosis factor-alpha (TNFα) and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were also measured. Treatment with LAB prevented vascular leakage and basement membrane thickening in retinal capillaries in a dose-dependent manner. Insulin resistance and glucose intolerance were significantly improved by LAB treatment. The levels of serum hsCRP, MCP1, TNFα, and urinary 8-OHdG were lower in the LAB-treated OLETF rats than in the controls. Conclusions Treatment with LAB had a preventive effect on the development of diabetic retinopathy in this animal model, probably because of its antioxidative effects and anti-inflammatory effects.
    PLoS ONE 06/2014; 9(6):e98232. DOI:10.1371/journal.pone.0098232 · 3.23 Impact Factor
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    • "In pulmonary fibrosis Erk5 also appears to be necessary for the production of ECM by fibroblasts and epithelial cells, however, in this case it is associated with Smad3 acetylation; acetylation of Lys19 on Smad3 enhances the binding of Smad3 to DNA (Kim et al., 2013). The activation of Erk5 by glucose in retinal and vascular endothelial cells causes a significant rise in fibronectin expression, a feature of diabetic retinopathy (Wu et al., 2012). Interestingly, in these experiments the effect of Erk5 may be Smad-independent as expression of constitutively active Erk5 was associated with a decrease in Smad2 phosphorylation. "
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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. DOI:10.3389/fphar.2014.00071 · 3.80 Impact Factor
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    ABSTRACT: Recent research using a rat oxygen-induced retinopathy model has demonstrated that the G protein-coupled receptor 91 (GPR91) of retinal ganglion neurons is the principal respondent to succinate and consequently induces the release of angiogenic factor vascular endothelial growth factor (VEGF). The aim of this study was to determine whether GPR91 modulate the release of VEGF from retinal ganglion cells in a high-glucose model in vitro and to dissect the role of GPR91 in the pathogenesis of diabetic retinopathy. We constructed a lentiviral small hairpin RNA (shRNA) expression vector targeting GPR91 (LV.shGPR91) and transfected the retinal ganglion cell line RGC-5 to obtain stably transfected cells. The knockdown effect of GPR91 was detected by Western blotting. After incubation with succinate and various concentrations of glucose, the expression of VEGF in RGC-5 cells was evaluated by real-time PCR and Western blotting, and the release of VEGF protein was measured using an ELISA assay. Conditioned media were also collected, and the effects of proliferation and migration of RF/6A cells, a vascular endothelial cell line, were evaluated by CCK8 and Transwell assays. The phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK) in RGC-5 cells after exposure to high glucose were evaluated by Western blotting. Following a single exposure of RGC-5 cells to the encoding lentivirus, more than 80% of transfectants expressed GFP at 72 h, and the level of GPR91 protein was significantly downregulated. GPR91 shRNA inhibited the cell survival rates of RGC-5 cells incubated with high glucose (F=21.36, P=0.002). The mRNA and protein expression of VEGF in LV.shGPR91 RGC-5 cells decreased markedly compared to that of LV.shScrambled or untransfected control cells incubated with different concentrations of glucose or succinate (P<0.01). The VEGF protein level in medium from RGC-5 cells treated with high glucose (F=57.43, P=0.000) or succinate (F=241.91, P=0.000) was also downregulated when transfected with GPR91 shRNA. The siRNA-mediated knockdown of GPR91 was also found to inhibit the proliferation of RF/6A cells in high glucose-stimulated (t=8.21, P=0.001) or succinate-stimulated (t=3.36, P=0.028) conditioned media. However, the siRNA-mediated knockdown of GPR91 suppressed the migration of RF/6A cells incubated with moderate levels of glucose (t=2.97, P=0.018). The exposure of RGC-5 cells to high glucose activated ERK1/2 and JNK MAPK signaling blocking by GPR91 shRNA (P<0.01). These results indicate that GPR91 modulates the high glucose-induced VEGF release of RGC-5 cells, possibly by inhibiting ERK1/2 and JNK MAPK signaling.
    Experimental Eye Research 02/2013; 109. DOI:10.1016/j.exer.2013.01.011 · 2.71 Impact Factor
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