Abstract
Connective tissue growth factor (CTGF) plays an essential role in the pathogenesis of diabetic nephropathy and we have previously identified that high glucose induced the expression of CTGF by decreasing DNA methylation. The aim of the present study was to investigate the underlying mechanisms of the high glucose-induced CTGF hypo-methylation. Human glomerular mesangial cells (hMSCs) were treated with low glucose (5 mM), mannitol (30 mM) or high glucose (30 mM) respectively. Immunofluorescence staining, real-time quantitative PCR and western blotting were performed to determine the subcellular distribution and expression of CTGF and Dnmt3a. ChIP-PCR assay was applied to investigate the capability of Dnmt3a to bind the CpG island of CTGF. Our results showed that high glucose induced both mRNA and protein expressions of CTGF, and led to increased cytoplasmic translocation of Dnmt3a in cultured hMSCs. The nuclear Dnmt3a protein was significantly reduced after high glucose treatment, although the expression of total Dnmt3a protein was not altered. We further discovered that ERK/MAPK signalling contributed to the high glucose-induced cytoplasmic translocation of Dnmt3a. Consequently, less Dnmt3a protein was bound to the CpG island of CTGF promoter, which induced an increase in CTGF expression by epigenetic regulation in the presence of high glucose. In conclusion, high glucose induces cytoplasmic translocation of Dnmt3a, possibly through activating ERK/MAPK signalling pathway, which contributes to the decreased binding of Dnmt3a on CTGF promoter and the subsequent CTGF hypo-methylation in diabetic nephropathy.
Highlights
Diabetic nephropathy (DN) is a major microvascular complication among diabetic patients and the most common cause of end-stage renal disease (ESRD), affecting around 30 % of type 1 and type 2 diabetic patients [1]
Compared with 0 h, total Dnmt3a expression did not change at 12, 24, 48 or 72 h post highglucose stimulus (Figure 2C). These results suggested that high glucose promoted cytoplasmic translocation of Dnmt3a, which might lead to the hypo-methylation [20] and the increased expression of Connective tissue growth factor (CTGF) (Figures 1A and 1B)
Our results demonstrated that increased cytoplasmic Dnmt3a and CTGF induced by high glucose was reversed when phosphorylated ERK (pERK) was inhibited by PD98059 (Figures 4B and 4C)
Summary
Diabetic nephropathy (DN) is a major microvascular complication among diabetic patients and the most common cause of end-stage renal disease (ESRD), affecting around 30 % of type 1 and type 2 diabetic patients [1]. Connective tissue growth factor (CTGF), a cysteine-rich protein with molecular weight around 38 kDa, promotes mesangial extracellular matrix synthesis, cellular hypertrophy, cell adhesion and mesangial matrix expansion [5,6] It is well known as a key factor in the pathogenesis and development of diabetic nephropathy. Up-regulation of CTGF mRNA and protein expressions has been confirmed in high glucose-induced mesangial cells and podocytes [7,8] in both diabetic nephropathy animal models [9,10], and type 1 and type 2 diabetic patients [11,12] This upregulation is highly correlated with the severity of albuminuria and the stage of renal insufficiency. It has been reported that specific blocking of CTGF by antisense oligonucleotide (ASO) significantly decreased proteinuria, reversed mesangial expansion in diabetic mice [13,14] and improved the attenuation of albuminuria in patients with microalbuminuric diabetic kidney
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