The Molecular Mechanisms Of Prohibitin Involving In Tubulointerstitial Fibrosis

BackgroundRenal interstitial fibrosis (RIF), accumulation of extracellular matrix (ECM) proteins in the renal interstitium, determines onset and progression of chronic renal failure in most renal diseases. RIF is a common character in end-stage renal diseases. Earlier work has demonstrated that transforming growth factor-β1 (TGF-β1) is the single most important fibrogenesis-inducing and propagating cytokine. Elevated TGF-β1 expression was noted in renal fíbrosis animal models and in patients with glomerulonephritis, diabetic nephropathy, and chronic allograft rejection. Inhibition of endogenous TGF-β1 activity in experimental fibrogenic renal disease models such as with neutralizing anti-TGF-β1 antibodies substantially reduces the development of renal fibrosis. In addition, mice lacking functional smad3 are partially protected from fíbrosis when challenged by induction of unilateral ureteral obstruction (UUO). Renal fibroblasts are thought to be the primary target of TGF-β1 and are the principal origin of interstitial ECM proteins. Very few fibroblasts and myofibroblasts normally are found in the kidney. However, in fibrogenic renal diseases, the number of fibroblasts increases by growth and they undergo a maturation and activation process that includes the expression of motor proteins such as myosin andα-smooth muscle actin (α-SMA).Although models of RIF have been developed both in rats and mice to examine these fibrosis mechanisms, the exactly molecular mechanisms remain largely unknown. Huang et al showed recently that, prohibitin (PHB), a protein with cell-cycle regulatory activity, was down-regulated by 50% in renal tissue of RIF rats induced by UUO with gene expression microarray analysis. PHB is a potential tumor suppressor which ubiquitously expressed in the mitochondria and in the nucleus of certain cell types. Current data support the dual function of PHB, first as a mitochondrial chaperone protein in a complex with prohibitone (also called PHB2) , and second as a regulator of cell cycle. Recent observations that PHB can interact with members of the E2F transcription factor family support the concept of a nuclear role in cell-cycle regulation. It has been demonstrated, using transfection assays, that PHB can reduce the amount of E2F1 mediated transcription by interacting directly with E2F1, as well as recruiting Rb, histone deacetylases and the corepressor NCoR. Up to date, most studies are focus on the role of PHB in some kinds of tumors while little is known about the role of PHB involved in RIF. In this study, we investigated the expression of PHB in human renal biopsy with different degree of tubulointerstitial lesions, in normal rat kidney fibroblasts (NRK-49F). We investigated the effects of PHB on cell growth and phenotypic change and ECM expression induced by TGF-β1 in NRK-49F cells. Then we ascertained whether PHB can suppress TGF-β1-mediated Smad signaling passway, and if so, how PHB antagonizes the profibrotic action of TGF-β1 in fibroblasts, aimed to illuminate the role of PHB in RIF.PartⅠThe expression of prohibitin in renal tissue of children with tubulointerstitial lesionsobjectives: To investigate PHB protein expression in human renal biopsy with different degrees of tubulointerstitial lesions, and to study the correlation among PHB expression, the degrees of tubulointerstitial lesions and renal function.Methods: Forty-eight specimens were obtained by percutaneous renal biopsy from patients with various primary glomerulonephritis, including 9 with MCD, 5 with FSGS, 4 with MN, 12 with MsPGN and 18 with IgA nephropathy, and nine normal kidney tissues were taken as normal control. According to tubulointerstitial injury under light microscopy, specimens were classified into four degree groups, with 23 cases in Grade 0 group, 13 in GradeⅠgroup, 12 in GradeⅡgroup and 10 in GradeⅢgroup. The expression of PHB andα-smooth muscle actin(α-SMA) protein were detected with immunohistochemical method, and interstitial fibrotic index was evaluated by Masson's trichrome staining respectively. The correlation among PHB,α-SMA, the degree of tubulointerstitial lesions and renal function were compared.Results: PHB protein expression was found at normal renal tissues by immunohistochemistry, with an obvious positive distribution in tubular epithelial cells and some interstitial cells. PHB was strongly down-regulated in damaged interstitial and tubular epithelial cells (between groups, F=82．6, P< 0．01) , which was negatively correlated with the degree of tubulointerstitial lesions and urinary N-acety1-β-D-glucosamccharase (NAG) (r=-0．802 and-0．713, Ps<0．05) . Expression of α-SMA was scanty in uninjured interstitial cells except vascular smooth muscle cells , but it was strongly up-regulated in damaged interstitial and tubular epithelial cells. Expression ofα-SMA was negatively correlated with the expression of PHB (r=-0．756, P=0．011) , but positively correlated with the degree of tubulointerstitial lesions(r=0．765, P=0．006) .Conclusions : PHB may be involved in the progression of tubulointerstitial lesions and down-regulation of PHB may be prognostic in renal diseases.PartⅡProhibitin suppresses renal interstitial fibroblasts proliferation, extra-cellularmatrix expression, and phenotypic change induced by transforming growthfactor-β1Objectives To investigate the expression of PHB in NRK-49F cells and investigatethe effects of PHB on cell growth, phenotypic change and ECM expression inducedby TGF-β1 in NRK-49F, aimed to illuminate the role of PHB in RIF.Methods (1) Subcellular location of PHB protein in NRK-49F cells was detected bylaser scanning confocal microscope. The changes of PHB protein and mRNAexpression in NRK-49F cells upon TGF-β1 stimulation were detected by Western blotand Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)analysis. (2) PHB expression plasmid was constructed and transfected into NRK-49Fcells. (3) Cell cycle analysis of NRK-49F cells was performed by flow cytometry, andgrowth curves were detected by MTT analysis. (4) PHB,α-SMA, ECM proteinfibronectin (Fn) and collagen typeⅢ(ColⅢ), and matrixmetalloproteinase-1(MMP-1) protein and mRNA expression in NRK-49F cell treatedwith or without TGF-β1 were detected by Western blot and RT-PCR analysisrespectively.Results (1) Confocal microscopy showed PHB is majority located at cytoplasm aswell as at nucleus in NRK-49F cells. PHB protein and mRNA expression in NRK-49Fcells were decreased when treated with TGF-β1, and the effects were bothtime-dependent and dose-dependent. (2) Recombinant pcDNA3．1(-) /PHB plasmidwas successfully constructed. The plasmid was sequenced by Bioasia company andthe nucleic acid sequence of 887 kb fragment was just the same as rat PHB(BC097304) recorded in GenBank. PHB protein expression in transfected NRK-49Fcells was 2．54 fold increased compared with non-transfected cells. (3) Stimulation of NRK-49F cells with 1ng/ml TGF-β1 resulted in a shift of cell cycle from G0/G1 to S and G2/M, with approximately 20% increase in the cell population activity in cell cycle. The over-expression of PHB did not appear to alter dramatically the number of cells entering into cell cycle without stimulation although there was a small drop in the number of cells in S and G2/M. In comparison, upon stimulation with TGF-β1, the non-transfected cells entered into the cell cycle as described above, whereas the PHB over-expressing cells failed to enter the cell cycle, with 90% of the transfected population remaining in G1 compared to approximately 73% of untransfected cells. By MTT assay, we found that, stimulation of cells with lng/ml TGF-β1 induced a remarkable cells growth. Transfected with PHB gene suppressed cells growth, and the cell growth was 27%, 48% and 35% inhibited after transfection for 24, 48 and 72 hours, respectively. Over-expression of PHB did not appear to alter dramatically the growth curve of cells without stimulation (5) Bothα-SMA protein and mRNA were not expressed in control cells while de novo expression ofα-SMA in NRK-49F cells was increased after the treatment of TGF-β1．Over-expression of PHB did not affect basicα-SMA expression but dramatically repressed TGF-β1-initiatedα-SMA expression in NRK-49F cells (Ps < 0．01) . We found that TGF-β1 induced ColⅢand Fn proteins and mRNA expression and the effects were dose-dependent. The expression of ColⅢ, Fn, and MMP1 protein were up-regulated 2．14, 1．68, and 1．87 folds, and expression of ColⅢ, Fn, and MMP1 mRNA were up-regulated 2．04, 1．80, and 1．77 folds in NRK-49F cells treated with lng/ml TGF-β1 for 48 hours compared with control cells. Over-expression of PHB had no effects on basic expression of these proteins and mRNA. While over-expression of PHB substantially attenuated the up-regulation of ColⅢand Fn, it had almost no effect on the up-regulation of MMP1 induced by TGF-β1 in NRK-49F cells.Conclusions: Extraneous PHB suppresses renal interstitial fibroblasts proliferation, cell phenotypic change and ECM expression induced by TGF-β1, which indicates PHB as a potential therapeutic target to halt the progression of TIF.PartⅢProhibitin suppresses profibrotic cytokine transforming growth factor-β1(TGF-β1) by intercepting Smad3 nuclear translocationObjectives To ascertain whether PHB can suppress TGF-β1-mediated Smadsignaling passway, and if so, how PHB antagonizes the prof?brotic action of TGF-β1 in fibroblasts, aimed to illuminate further the role of PHB in RIF.Methods NRK-49F cells transfected or non-transfected with pcDNA3．1(-) /PHB plasmid were starved by exposure to serum-free medium for 48 hours, and then treated with or without 1ng/ml TGF-β1 for another 30 min or 48 hours as indicated before harvesting. Then we examined the phosphorylation and activation of Smad3 by TGF-β1 and Smad7 expression in cells by Western blot using specific p-Smad3 and Smad7 antibody, and further investigated the phosphorylation of Smad3 and its association with Smad4 in cells after TGF-β1 stimulation by co-immunoprecipitation. Cell nuclei were isolated from cells after various treatments as indicated and nuclear accumulation of p-Smad3 was examined by Western blot. The samples were also probed with ubiquitous transcription factor Sp1 for normalization of nuclear proteins.Results Smad3 was phosphorylated after TGF-β1 treatment, as demonstrated by the presence of phosphoserine-specifìc bands in the complexes immunoprecipitated by anti-Smad2/3 antibody. Co-immunoprecipitation also revealed that p-Smad3 was physically associated with Smad4 after TGF-β1 stimulation. However, Over-expression of PHB did not significantly affect Smad3 phosphorylation and its association with Smad4．Neither TGF-β1 nor PHB significantly affect Smad7 protein expression after incubating with TGF-β1 for 48 hours.Conclusions The inhibitory effect of PHB on both TGF-β1-stimulated phenotypic changes and ECM proteins expression may primarily by preventing activated Smad3 nuclear translocation and accumulation, which suggest that PHB has potential anti-fibrotic effects. Therefore strategies to restore or increase PHB expression level may constitute a novel therapeutic approach for halt the progression of RIF.Conclusions1,Here, for the first time, we reported that PHB protein was expressed at normal human renal tissues, with an obvious distribution in interstitial cells and tubular epithelial cells and adversely correlated with the degree of tubulointerstitial lesions..2,Confocal microscopy showed PHB is majority located at cytoplasm as well as at nucleus in rat kidney fibroblasts cell (NRK-49F). PHB protein and mRNA expression in NRK-49F cells were decreased when treated with TGF-β1, and the effects were both time-dependent and dose-dependent.3,Extraneous PHB suppresses renal interstitial fibroblasts proliferation, cell phenotypic change and ECM expression induced by TGF-β1．4,Prohibitin suppresses profibrotic cytokine transforming growth factor-β1 (TGF-β1) by intercepting Smad3 nuclear translocation, which indicates PHB as a potential therapeutic target to halt the progression of TIF.