Prohibitin Protein Expression Research Articles

Prohibitin Protein Expression During Spermatogenesis in the Large Yellow Croaker, Larimichthys crocea

Prohibitin Protein Expression During Spermatogenesis in the Large Yellow Croaker, Larimichthys crocea

Emodin Improves Glucose and Lipid Metabolism Disorders in Obese Mice via Activating Brown Adipose Tissue and Inducing Browning of White Adipose Tissue.

BackgroundAdipose tissue (e.g. white, brown and brite) plays a critical role in modulating energy metabolism. Activating brown adipose tissue (BAT) and inducing browning in white adipose tissue (WAT) has been proposed to be a potential molecular target for obesity treatment. Emodin is a natural anthraquinone derivative that exhibits variety of pharmacologic effects including lowering lipids and regulating glucose utilization. However, the underlying mechanism of action is still unclear. In the present study, we investigated whether emodin could alleviate obesity via promoting browning process in adipose tissue.MethodsC57BL/6J mice were fed with high fat diet to induce obesity. Emodin at the doses of 40 and 80 mg/kg were orally given to obesity mice for consecutive 6 weeks. Parameters including fasting blood glucose, oral glucose tolerance, blood lipids, and the ratios of subcutaneous white adipose tissue (scWAT) or BAT mass to body weight, and morphology of adipose tissue were observed. Besides, the protein expression of uncoupling protein 1 (UCP1) and prohibitin in BAT and scWAT was determined by immunohistochemistry method. Relative mRNA expression of Cd137, transmembrane protein 26 (Tmem26) and Tbx1 in scWAT was analyzed using qRT-PCR. And the protein expression of UCP1, CD36, fatty acid transporter 4 (FATP4), peroxisome proliferator-activated receptor alpha (PPARα) and prohibitin of scWAT and BAT were analyzed using western blotting. In addition, ultra-high-performance liquid chromatography with electrospray ionization tandem mass spectrometry was utilized to detect the small lipid metabolites of scWAT and BAT.ResultsEmodin decreased the body weight and food intake in HFD-induced obesity mice, and it also improved the glucose tolerance and reduced the blood lipids. Emodin treatment induced beiging of WAT, and more multilocular lipid droplets were found in scWAT. Also, emodin significantly increased markers of beige adipocytes, e.g. Cd137, Tmem26 and Tbx1 mRNA in scWAT, and UCP1, CD36, FATP4, PPARα and prohibitin protein expression in scWAT and BAT. Furthermore, emodin perturbed the lipidomic profiles in scWAT and BAT of obese mice. Emodin increased total ceramides (Cers), lysophosphatidylcholines (LPCs), lyso-phosphatidylcholines oxygen (LPCs-O), and phosphatidylethanolamines oxygen (PEs-O) species concentration in scWAT. Specifically, emodin significantly up-regulated levels of Cer (34:1), LPC (18:2), LPC-(O-20:2), PC (O-40:7), PE (O-36:3), PE (O-38:6), PE (O-40:6), and sphingolipid (41:0) [SM (41:0)], and down-regulated PC (O-38:0), PE (O-40:4), PE (O-40:5) in scWAT of obesity mice. In terms of lipid matabolites of BAT, the emodin remarkably increased the total PCs levels, which was driven by significant increase of PC (30:0), PC (32:1), PC (32:2), PC (33:4) and PC (38:0) species. In addition, it also increased species of LPCs, e.g. LPC (20:0), LPC (20:1), LPC (22:0), LPC (22:1), LPC (24:0), and LPC (24:1). Especially, emodin treatment could reverse the ratio of PC/PE in HFD-induced obese mice.ConclusionsThese results indicated that emodin could ameliorate adiposity and improve metabolic disorders in obese mice. Also, emodin could promote browning in scWAT and activate the BAT activities. In addition, emodin treatment-induced changes to the scWAT and BAT lipidome were highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflects selective remodeling in scWAT and BAT of both glycerophospholipids and sphingolipids in response to emodin treatment.

Characterization of PHB in the gonadal development of the swimming crab Portunus trituberculatus.

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. In this study, we cloned the PHB gene from the testis of the swimming crab Portunus trituberculatus (PtPHB) and analyzed the deduced amino acid sequence. The expression level of phb mRNA in larvae was analyzed using qRT-PCR. The expression level of phb mRNA and PHB protein in different tissues were analyzed using qRT-PCR and Western blot respectively. Enzyme-linked immunosorbent assay analyses of the PHB protein were conducted with the testis and ovaries from P. trituberculatus specimens at different developmental stages. PHB was localized with mitochondria and ubiquitin in the testis and ovaries. The PtPHB gene was found to contain an open reading frame of 825 bp, encoding a predicted peptide with 275 amino acids, sharing between 65.9% and 96.7% similarity with that of other species. The qRT-PCR and Western blot results showed that the phb gene and PHB protein both expressed less in the testis and ovary than in other tissues, and the phb gene presented the lowest expression in the Z1 stage. Furthermore, the phb gene and PHB protein expression were different in the testis and ovaries at different developmental stages. PHB was mainly found to be co-localized with mitochondria and ubiquitin in cytoplasm and acrosome complex during spermatogenesis and in follicular cells during oogenesis. Interestingly, PHB-mitochondria signals and ubiquitin signal were also found in oocytes. These results indicated that PHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities.

The mechanism of overexpression of retinoic acid receptor alpha in attenuating renal interstitial fibrosis in rats

Objective To study the mechanism of overexpression of retinoic acid receptor alpha (RARα) in attenuating renal interstitial fibrosis (RIF) in rats. Methods Forty 6-week-old male SD rats were randomly divided into 4 groups: sham operation group, model group, negative control group and transfection group, with 10 rats in each group.Rats in model group were separated and double ligated with left ureter; rats in sham operation group were not ligated with ureter; rats in transfection group and negative control group were transfected with adeno-associated virus and negative control virus carrying RARα gene on the basis of model group, respectively.All rats were sacrificed 2 weeks later.Left kidney tissues were taken for pathological examination and RIF index was calculated.The expression of collagen Ⅳ (Col-Ⅳ) and fibronectin (FN) in renal tissue was detected by using immunohistochemistry.The expressions of RARα, prohibitin (PHB) and transforming growth factor-beta 1 (TGF-β1) in renal tissue were detected by using real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot. Results (1)Compared with sham operation group, the RIF index was significantly increased in model group (22.81±2.43 vs.2.34±0.55, q=24.94, P 0.05), however it significantly decreased in transfection group (14.06±2.99 vs.22.81±2.43, q=10.66, P 0.05); compared with model group, the mRNA and protein expression of RARα, PHB mRNA and protein expression significantly increased, but the TGF-β1 mRNA and protein, Col-Ⅳ and FN protein expression significantly decreased in transfected group (mRNA: 0.86±0.07 vs.0.43±0.17, 0.89±0.11 vs.0.34±0.08, 1.65±0.28 vs.2.97±0.54, all P<0.05; protein: 0.40±0.07 vs.0.25±0.10, 0.45±0.10 vs.0.24±0.07, 0.43±0.08 vs.0.59±0.09, 11.57±0.33 vs.16.01±0.87, 11.67±0.53 vs.14.64±0.32, all P<0.05). (3)Correlation analysis revealed that RARα protein expression was negatively correlated with RIF index, Col-Ⅳ, FN, TGF-β1 (r=-0.78, -0.78, -0.76, -0.76, all P<0.05); PHB protein expression was negatively correlated with RIF index, Col-Ⅳ, FN, TGF-β1 (r=-0.87, -0.87, -0.88, -0.75, all P<0.05); RARα protein was positively correlated with PHB (r=0.85, P<0.05). Conclusion Overexpression of RARα could attenuate RIF by enhancing PHB expression in rats subjected to unilateral ureteral obstruction. Key words: Retinoic acid receptor alpha; Prohibitin; Renal interstitial fibrosis; Transfection

Diagnostic and Prognostic Implications of Prohibitin Overexpression in Normal Karyotype Acute Myeloid Leukemia

Abstract Background: Prohibitin (PHB) is a highly conserved protein, widely expressed in multiple cellular compartments such as mitochondria and nucleus. Recent studies have demonstrated that PHB played an important role in the regulation of mitochondrial function, cell proliferation and development. Our proteome analysis has revealed PHB was highly expressed in primary AML cells compared with normal cells. This finding prompted us to investigate whether PHB could serve as a potential diagnostic, prognostic, and therapeutic target in AML. Patients and Methods: PHB2 protein expression was assessed in 133 newly diagnosed normal karyotype AML (NK-AML) patients by using immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections. The degree of PHB2 protein expression was scored as 0-8 as a sum of a diffuseness score (0-5) and an intensity score (0-3). Overall survival (OS) and event-free survival (EFS) was analyzed according to PHB2 expression score. Also, we developed quantitative reverse transcriptase PCR (qRT-PCR) method to measure mRNA expression of PHB1 and PHB2 genes for the optimal algorithms for the discrimination of AML cells from the normal blood or hematopoietic cells. Because accumulation of beta-catenin in the nucleus has been identified in leukemic cells, we investigated the effect of beta-catenin accumulation on PHB expression. We monitored the expression levels of PHB with lithium chloride treatment, which blocks GSK-3beta activity and ultimately affects beta-catenin accumulation. For the development of anti-leukemic drug candidates targeting PHB in AML cells, we synthesized two potent chemical substances that can alkylate PHB: cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). We evaluated their therapeutic effects in the primary AML and AML cell lines. Results: In the PHB2 IHCS, using the cutoff as positivity score 4.0, PHB2 positive patients showed inferior OS (23.7% vs 50.0%; P = 0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 negative patients (figure 1). Using the same cutoff, PHB2 positive patients showed inferior EFS (20.7% vs 47.1%; P= 0.024) and high hazard ratio (1.795; P = 0.026) (figure 2). In the qRT-PCR assay of PHB1 and PHB2, quantitative expression (ddCt) of PHB1 gene was higher in the control group: 0.31-28.32 (4.40+/-5.80; Mean+/-SD) versus 0.22-6.52 (1.83+/-1.56) in patient group. Meanwhile, PHB2 gene expression was higher in the NK-AML patient group: 0.15-3.49 (0.80+/-0.47) versus 0.00-0.46 (0.16+/-0.13) in control group. Using linear discrimination analysis, we derived a linear equation for discrimination between normal and AML cells. Estimating the prevalence of AML as 57.2/1,000,000, the sensitivity of newly developed algorithm was 93.6%, specificity was 76.9%, negative predictive value was 99.9%, and positive predictive value was 2.28% (figure 3). PHB protein expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This beta-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicated that PHB was a direct target of beta-catenin and the increased level of PHB in leukemic cells can be regulated by Wnt signaling. Using CCEU and ICEU, time and dose dependent manner of proliferation suppression was observed in primary AML and AML cell lines. Notable morphological transformation of AML cells was observed when treated with 10-100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25 umol for most AML cells. Conclusions: Overexpression of PHB2 was associated with poor prognosis in NK-AML. Newly developed discrimination algorithm using qRT-PCR showed the possibility as a rapid diagnostic tool for leukemic cell identification. Our functional study suggested that the elevated level of PHB in leukemic cells was the result of Wnt signals which were up-regulated in AML cells. Finally, this study developed novel alkylating chemotherapeutics targeting PHB for selective killing of leukemic cells. PHB2 expression status can provide clinically valuable information for the diagnosis, prediction of the prognosis, detection of minimal residual disease, and therapeutic targeting of NK-AML. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

The change of prohibitin in the kidney injury of db/db mice and its intervention

Objective To study the molecular mechanisms of prohibitin in the diabetic nephropathy and its effect. Methods C57BLKS/J db/db mice were selected randomly as diabetic model group (DM group, n=8) and the db/m mice were selected as control group (CC, n=8), and C57BLKS/J db/db mice lavage treated with GSPB2 for 10 weeks were considered as treatment group (30mg/kg body weight/day, DMT, n=8). At the end of 18 weeks, fasting blood glucose (FBG), advanced glycation end products (AGEs) and monocyte chemotactic protein 1 (MCP1) were determined. Morphological changes of the kidney tissue were examined by light microscopy and transmission electron microscope. The expression of prohibitin in the kidney tissue was determined by Western blot. Results The body weight was higher in DM groups than in control group at 8, 12, 16, 18 weeks (all P<0.01). The body weight was significantly inhibited at 12, 16, 18 weeks after GSPB2 administration in the DMT group as compared to the DM group (all P<0.05). Serum FBG, AGEs and MCP-1 levels were significantly higher in DM group than in control group (all P<0.01). GSPB2 significantly decreased the serum levels of FBG, AGEs and MCP-1 in db/db mice (all P<0.01). The glomerular volume, mesangial cell proliferation, extracellular matrix in DM group was significantly increased as compared with CC group by light microscopy. GSPB2 significantly reduced the glomerular volume, mesangial cell proliferation in db/db mice. The result of ultrastructural microscopy showed that in DM group, mesangial cell was inserted into the sub-endothelium, basement membrane became thicken, the number of podocytes was decreased and the podocytes were fused, and the thicken basement membrane and podocytes injure were improved in the DMT group. Moreover, the protein expression of prohibitin was higher in DM group than in control group (P<0.01), while GSPB2 could decrease the protein expression of prohibitin (P<0.01). Conclusions The mechanism of protective effects of GSPB2in diabetic nephropathy can be attributed to its anti-glycation, anti-inflammatory and the increase of prohibitin expression, which will provide a theoretical basis for the prevention and treatment of diabetic nephropathy. Key words: Diabetic nephropathy; Ceu prohibitin; Procyanidins

Immunohistochemical expression and mutation study of Prohibitin gene in Indian female breast cancer cases

Prohibitin (PHB1) is highly conserved and ubiquitously expressed protein. It is mapped to the chromosome 17q12-q21 locus, a region that has been reported to be genetically linked to early onset of breast cancer. Therefore, we carried out the population-based study in a total of 105 Indian female breast cancer cases and analyzed mutation(s) on exon 4 and the introns flanking it. Importantly, it has been found that the region of this exon has specific binding site for Rb and p53 gene. We further did protein expression of Prohibitin through immunohistochemistry in the same set of population where mutation has already been found. Out of 105 breast cancer cases, 46 cases (46/105, 43.8%) showed low or no expression (+), 19 cases (19/105, 18.0%) with moderate (++) expression and 40 cases (40/105, 38.0%) had high (+++) expression for Prohibitin. Highly significant association was observed statistically between Prohibitin protein expression and clinico-pathological variables like nodal status (p=0.0003), tumor stage (p=0.0001), histological grade (p=0.009). Moreover, the previously found mutation(s) when analyzed with the immunohistochemistry data revealed that all the breast cancer cases with mutation representing intron deletion (deletion of T nucleotide) near the intron-exon boundary had low (+) or no expression for Prohibitin. In summary, Prohibitin may be associated with breast cancer and its down expression can serve as a potential biomarker for the effective assessment of the disease.

Less Expression of Prohibitin Is Associated with Increased Paired Box 2 (PAX2) in Renal Interstitial Fibrosis Rats

Prohibitin (PHB) and paired box 2 (PAX2) are associated with the development of renal interstitial fibrosis (RIF). This study was performed to investigate whether or not the PHB could regulate the PAX2 gene expression in unilateral ureteral obstruction (UUO) in rats. Eighty Wistar male rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 40, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14-day and 28-day after surgery. RIF index, protein expression of PHB, PAX2, transforming growth factor-βl (TGF-β1), α-smooth muscle actin (α-SMA), collagen-IV (Col-IV), fibronectin (FN) or cleaved Caspase-3, and cell apoptosis index in renal interstitium, and mRNA expressions of PHB, PAX2 and TGF-β1 in renal tissue were detected. When compared with those in SHO group, expression of PHB (mRNA and protein) was significantly reduced, and expressions of PAX2 and TGF-β1 (protein and mRNA) were markedly increased in the GU group (each p < 0.01). Protein expressions of α-SMA, Col-IV, FN and cleaved Caspase-3, and RIF index or cell apoptosis index in the GU group were markedly increased when compared with those in the SHO group (each p < 0.01). The protein expression of PHB was negatively correlated with protein expression of PAX2, TGF-β1, α-SMA, Col-IV, FN or cleaved Caspase-3, and RIF index or cell apoptosis index (all p < 0.01). In conclusion, less expression of PHB is associated with increased PAX2 gene expression and RIF index in UUO rats, suggesting that increasing the PHB expression is a potential therapeutic target for prevention of RIF.

Vitamin D deficiency decreases the expression of VDR and prohibitin in the lungs of mice with allergic airway inflammation

AimsAsthma is one of the most common chronic inflammatory diseases of the airways. Calcitriol exerts its action through Vitamin D receptor (VDR), which is a high affinity nuclear receptor. VDR is a transcription factor that alters the transcription of target genes which are involved in a wide spectrum of biological responses. Lower serum vitamin D levels are associated with airway hyperresponsiveness and increased asthma severity. Prohibitin is a ubiquitously expressed protein localized to the cell and mitochondrial membranes and the nucleus. Methods and resultsHBSMCs were cultured and treated with calcitriol and/or TNF-α. The mRNA and protein expression of prohibitin and VDR were analyzed using qPCR and immunoblotting, respectively. In the in vivo studies, female BALB/c mice were fed with special vitamin D-deficient or 2000IU/kg of vitamin D-supplemented diet for 13weeks. Mouse model of allergic airway inflammation was developed by OVA-sensitization and challenge. The expression pattern of TNF-α, prohibitin and VDR in the lung of OVA-sensitized mice was analyzed using immunofluorescence. Calcitriol significantly increased and TNF-α decreased the protein and mRNA expression of prohibitin and VDR in HBSMCs. There was significantly increased expression of TNF-α and decreased expression of VDR and prohibitin in the lung of vitamin D-deficient mouse model of allergic airway inflammation. ConclusionThese results suggest that under inflammatory conditions there is decreased expression of VDR resulting in decreased expression of prohibitin, which is a vitamin D target gene. Vitamin D deficiency causes increase in the expression of TNF-α, thereby increasing inflammation and decreases the expression of VDR and prohibitin. Supplementation with vitamin D might reduce the levels of TNF-α, thereby increasing the expression of VDR and prohibitin that could be responsible for reducing allergic airway inflammation.

Emerging role of microRNA-27a in human malignant glioma cell survival via targeting of prohibitin.

MicroRNAs (miRs) function as oncogenes and tumor suppressors, and have roles in most cellular processes. To date, the possible role of miR-27a, which is part of the miR-23a/27a/24-2 cluster, in malignant gliomas has remained elusive. Therefore, the present study aimed to explore the role of miR-27a in glioma and its potential target. Through transfection with miR-27a inhibitor or oligonucleotide mimics, the impact of miR-27a silencing or overexpression on the growth, apoptosis, cell cycle and invasiveness of U251 and U87MG cells was examined in vitro. The present study initially identified the potential target of miR-27a in glioma cells through a bioinformatics analysis, which was used for screening of the literature on the proteomics of gliomas. Prohibitin (PHB) was then confirmed as a target by absolute luciferase reporter assays, quantitative real-time polymerase chain reaction and western blot analysis. Treatment with miR-27a mimics oligonucleotides suppressed U251 cell proliferation, promoted apoptosis by inducing G2/M phase arrest, and impaired the invasive potential of U251 cells in vitro. In addition, miR-27a expression was downregulated in glioma tissues. A PHB-3'-untranslated region luciferase reporter assay confirmed PHB as a direct target gene of miR-27a. PHB mRNA expression was reversely correlated with levels of miR-27a in U251 cells. Overexpression of miR-27a in U251 cells at 72 h and 96 h post‑transfection with miR-27a mimics significantly decreased PHB protein expression by 17.2% and 43.9%, respectively. In conclusion, miR-27a was shown to be a significant tumor suppressor by targeting and decreasing PHB protein expression in glioma U251 cells. miR-27a targeting of PHB may be a novel potential therapeutic strategy for glioma.

All-Trans Retinoic Acid Treatment Is Associated with Prohibitin Expression in Renal Interstitial Fibrosis Rats

This study was performed to investigate the association of prohibitin with renal interstitial fibrosis (RIF) lesion and to explore the association of all-trans retinoic acid (ATRA) treatment with prohibitin expression in RIF rats. Rats were divided into three groups: the sham operation group (SHO), the model group subjected to unilateral ureteral obstruction (UUO), and the model group treated with ATRA (GA). Renal tissues were collected at 14 and 28 days after surgery, and the relevant indicators were detected. In comparison with the SHO group, the RIF index in the UUO group was markedly elevated (p < 0.01), and the RIF index in the GA group was alleviated compared with that in the UUO group (p < 0.01). Compared with the SHO group, the expression of prohibitin (protein or mRNA) in the UUO group was significantly reduced (each p < 0.01). Prohibitin expression in the GA group was markedly increased when compared with that in the UUO (p < 0.01). The expression of TGF-β1 (protein and mRNA), protein expressions of Col-IV, fibronectin, α-SMA and cleaved Caspase-3, ROS generation and cell apoptosis index in the UUO group were markedly higher than those in the SHO group (all p < 0.01), and their expressions in the GA group were markedly down-regulated compared to those in the UUO group (all p < 0.01, respectively). The protein expression of prohibitin was negatively correlated with the RIF index, protein expression of TGF-β1, Col-IV, fibronectin, α-SMA or cleaved Caspase-3, ROS generation and the cell apoptosis index (each p < 0.01). In conclusion, lower expression of prohibitin is associated with the RIF, and ATRA treatment is associated with increased prohibitin, which can prevent the progression of RIF.

Prohibitin 1 Modulates Mitochondrial Stress-Related Autophagy in Human Colonic Epithelial Cells

IntroductionAutophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.MethodsWe measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.ResultsTNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.ConclusionsDecreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.

Less expression of prohibitin is associated with increased Caspase‐3 expression and cell apoptosis in renal interstitial fibrosis rats

Prohibitin (PHB), a ubiquitous protein, is involved in a variety of molecular functions. Renal interstitial fibrosis (RIF) is a hallmark of common progressive chronic diseases that lead to renal failure. This study was performed to investigate whether PHB was associated with caspase-3 expression/cell apoptosis in RIF rats. Twenty-four male Wistar rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 12, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14 days and 28 days after surgery. RIF index, cell apoptosis index, protein expression of PHB, transforming growth factor-βl (TGF-β1), collagen-IV (Col-IV), fibronectin (FN) or caspase-3 in renal interstitium, and mRNA expression of PHB in renal tissue were detected. Compared with that in the SHO group, the PHB expression (mRNA and protein) was significantly reduced (P < 0.01). Protein expressions of TGF-β1, Col-IV, FN and caspase-3, and RIF index or cell apoptosis index in GU group were markedly elevated compared with those in SHO group (all P < 0.01). The protein expression of PHB had a negative correlation with the protein expression of TGF-β1, Col-IV, FN or caspase-3, and RIF index or cell apoptosis index (each P < 0.01). Less expression of PHB is associated with increased caspase-3 expression/cell apoptosis in RIF rats. However, further research is needed to determine the effect of PHB on caspase-3 expression/cell apoptosis and to determine the potential of PHB as a therapeutic target.

Prohibitin is overexpressed in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length hepatitis C virus RNA

BackgroundCurrently, up-regulated proteins and apoptosis in hepatitis C is a hot topic in exploring the pathogenic mechanism of Heptitis C Virus(HCV). Some recent studies shows that prohibitin is overexpressed in cells expressing HCV core proteins, and up-regulated prohibitin is also found in human hepatoma cell line HCC-M, lung cancer, prostate cancer, and other cancers. Prohibitin is an important member of the membrane protein superfamily, and it plays a role of molecular chaperones in mitochondrial protein stability. Meanwhile, it has a permissive action on tumor growth or acts as an oncosuppressor. Based on our previously established the in vitro HCV cell-culture system (HCVcc), here we aimed to investigate the different expression profiles of prohibitin in Huh-7-HCV and Huh-7.5-HCV cellsMethodsThe total cellular RNA of Huh-7, Huh-7.5, Huh-7-HCV and Huh-7.5-HCV cells were extracted, and then the first-strand cDNA was reversely transcribed. The expression of prohibitin at the mRNA level was assessed by real-time PCR with GAPDH as the control. Furthermore, the expression of prohibitin at the protein level was evaluated by western blot with GAPDH as an internal control.ResultsOur results of real-time PCR showed that the mRNA expression level of prohibitin in Huh-7-HCV cells was 2.09 times higher than that in Huh-7 cells, while, the mRNA level of prohibitin in Huh-7.5-HCV cells was 2.25 times higher than that in Huh-7.5 cells. The results of western blot showed that the protein expression level of prohibitin in Huh-7-HCV cells was 2.38 times higher than that in Huh-7 cells, while the protein expression of prohibitin in Huh-7.5-HCV cells was 2.29 times higher than that in Huh-7.5 cells.ConclusionsThe expression of prohibitin was relatively high in Huh-7-HCV and Huh-7.5-HCV cells harboring in vitro transcribed full-length HCV RNA.

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1

To illuminate the possible role of Prohibitin (PHB) in tubulointerstitial fibrosis. (1) Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis, 26 male and 22 female, aged 7.5 +/- 5.5 (2.5 - 13 years), and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-SMA). The correlation between PHB and degree of tubulointerstitial lesion was compared. (2) Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. (1) PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB (all P < 0.01), and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions (r = -0.802, P < 0.01). (2) Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfection of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-SMA expression in the NRK-49F cells (P < 0.01). PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.

Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary.

Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but it increases during granulosa cell differentiation as well as in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In the present study, we have characterized the spatial expression patterns for prohibitin using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative Western blot analyses of granulosa cell lysates from control ovaries and from ovaries primed with eCG or treated with eCG plus anti-eCG (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed with antibodies against either proliferating cell nuclear antigen (PCNA) or cholesterol side-chain cleavage cytochrome P450 (P450(scc)) or in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled sections. Additionally, porcine oocytes, zygotes, and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells, and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and colocalized with P450(scc). Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes, and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.

Immunolocalization and expression of prohibitin, a mitochondrial associated protein within the rat ovaries.

This study was designed to determine the cellular distribution and pattern of expression for the mitochondria-associated protein, prohibitin, during the transitional stages of follicular differentiation within the rat ovary. Immunohistochemical staining techniques were used on frozen sections to examine the localization of prohibitin to preantral, antral, preovulatory, and atretic follicles. Prohibitin localization was also determined in corpus luteum from adult rats, in addition to those from infant and juvenile ovaries, before and after gonadotropin stimulation. Western and Northern blotting techniques were used for qualitative and quantitative assessment of prohibitin expression levels within the ovary. Prohibitin was localized within granulosa cells of infant and juvenile ovaries in a relatively heterogeneous staining pattern. The oocyte also exhibited robust prohibitin expression at all stages of follicular development. In addition, strong prohibitin expression was evident in the corpus luteum as well as in follicles undergoing atresia. Additional data derived from studies involving a GnRH-agonist indicate that increases in prohibitin protein expression correlate with the initial events of apoptosis. Collectively, these results support a growth regulatory role for prohibitin within the rat ovary. Therefore, we propose that prohibitin may serve as an important regulator of granulosa cell fate during follicular development.