Hrd1 participates in the regulation of collagen I synthesis in renal fibrosis

The production and accumulation of collagen-rich extracellular matrix are common hallmarks during the process of renal fibrogenesis. However, the mechanisms of the regulation of collagen synthesis in renal fibrosis are still unclear. Hrd1, an E3 ubiquitin ligase, plays important roles for protein folding in ER and transport to Golgi. Here, we examined the hypothesis that Hrd1 posttranslationally regulates collagen synthesis in renal interstitial fibrogenesis. Unilateral ureteral obstruction induced Hrd1 expression, predominantly in the renal interstitium and tubular epithelium of fibrotic kidneys. Transforming growth factor β1, as a key mediator in kidney fibrosis, significantly increased the expressions of Hrd1, α-smooth muscle actin, fibronectin as well as procollagen I and mature collagen I in dose-dependent manner in tubular epithelial cells, suggesting that collagen I maturation might be modulated during renal fibrosis. In cultured renal fibroblasts, Hrd1 knockdown decreased secreted collagen I ~60 % in the supernatant of NRK-49F cells. Conversely, Hrd1 overexpression increased secreted collagen I ~1.5-fold. Hrd1 overexpression significantly increased the expressions of both procollagen I and mature collagen I, ~2.2-fold and ~1.8-fold, respectively. However, Hrd1 knockdown markedly decreased the expression of mature collagen I ~80 %, while procollagen I expression only was decreased ~21 %. Moreover, short interfering RNA-induced knockdown of Sec23A blunted the increase in collagen I expression (both immature and mature form) by Hrd1 overexpression and returned collagen I expression toward control levels. These results indicate that Hrd1 plays an important role in the maturation of collagen I in renal fibrosis, and that Sec23A pathway is required for ER-to-Golgi procollagen trafficking to promote collagen synthesis.     

针对I型及Ⅲ型前胶原基因的二联核酶的构建及体外活性研究

增殖性瘢痕组织中胶原蛋白的合成显著增加从而导致胶原的过度沉积。利用核酶特异地抑制前胶原基因的表达可减少胶原蛋白的合成,为瘢痕的研究和防治提供了新的思路。为研究用核酶抑制前胶原基因表达的可能及效果,设计并构建了针对α1（I）型及α1（Ⅲ）型前胶原基因的二个单价核酶串联的二联核酶基因真核表达载体,并对其体外切割活性 进行研究。结果表明该二联核酶的切割效果明显,均能有效地切割底物,为进一步研究核酶的前胶原基因表达的抑制作用以及利用核酶防治瘢痕产生打下基础。     

Glucocorticoids coordinately regulate type I collagen proα1 promoter activity through both the glucocorticoid and transforming growth factor β response elements: A novel mechanism of glucocorticoid regulation of eukaryotic genes

Glucocorticoids have previously been shown to decrease Type 1 collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type 1 procollagen gene expression. These latter studies have included uridine incorporation into proα1(I) and proα2(1) mRNas and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking regionof the proα1 (1) coullagen gene and the reporter gene, chljoramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibrolblasts that dexamethasone down regulates the promoter activity of the proα1(I) collagen gene. The glucocorticoid-mediated down-regulastionof procolljagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whoulue ColCat 3.6 plasmid did not elimiinatre the effect. The ipossibility existed that another cis-element inthe 5' flanking region of the proα1(I) collagen gene was also required for the glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-β has been shown to stimulate collagen proα1(I) and proα2(I) gene activities. Dexamethasone treatment of non-transfected skin fibroblasts did result in a decrease of transforming growth factor-β. The decrease of CVAT activity by dexamethasone was brought back to control value by the addition of exogenous TGF-β to the culture media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid recptor binding to the GRE and TGF-β activator protein to the TGF-β element which were brought back to control values by coordinate exogenous TGF-β treatment. Thus the interaction of these TGF-β molecules with cellular membrane receptors and subsequent rtransduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expfrssion through both the GRE and TGF-β elements. Depression of procollagen gene expression by glucocorticoids through the TGF-β element is mediated by decreased TGF-β secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the bassis for a novel mechanism of glucocorticoid-mediated regulation of eukaryotic genes containing the TGF-β element. © 1995 Wiley-Liss, Inc.     

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.     

Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells

Although basic fibroblast growth factor (FGF-2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3-E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine-specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady-state mRNA levels of type I collagen. FGF-2 (30 ng/ml), platelet-derived growth factor-BB (PDGF-BB), 30 ng/ml, and serum, which activate ERK mitogen-activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3-E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c-fos induction by FGF-2 and PDGF-BB, suggesting that c-fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF-2 and PDGF-BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway.     

Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.