The Expression And Function Of RhoA/Rho-Kinase Pathway In Benign Prostatic Hyperplasia

Benign prostatic hyperplasia(BPH)is a common disease in older men,with a 50%incidence at 60 years old and up to 90%at 85 years old.BPH-induced lower urinary tract symptoms(LUTS)interfere with daily activities and sleep,mainly manifested as dysuria,urgency,frequent urination,nocturia increased and urinary incontinence,seriously affecting the quality of life.The pathogenesis of BPH is still not entirely clear.At present,BPH/LUTS on the one hand is due to bladder outlet obstruction and urethra caused by increased prostate volume,known as static factors;the other is caused by the prostate tension(neurogenic tension and spontaneous tension)and bladder overactivity,known as the dynamic factors,both play an important role in the development of BPH/LUTS.RhoA is a small guanosine triphosphatase,a member of the small G protein family,which plays an important role in the process of cell signal transduction.Rho-associated coiled-coil containing protein kinase(ROCK)is the most important downstream molecule of RhoA.The RhoA/ROCK pathway has been found involved in a variety of cellular functions,while its effect on smooth muscle contraction is widely studied.Some studies found that the expression of RhoA/ROCK pathway increased in the obstructed bladder and ureteral tissue,which lead to abnormal increase of tension.Recent studies have shown that the RhoA/ROCK pathway is expressed in the prostate tissue and is associated with prostate contraction.This suggests that the RhoA/ROCK pathway may be involved in the dynamic factors of BPH.Epithelial-mesenchymal transition(EMT)is known as epithelial cells transformed to mesenchymal cell phenotype induced by specific cytokines,which involved in embryonic development,organ fibrosis,cancer invasion and metastasis.Studies found that EMT involved in BPH,leading to the accumulation of stromal cells in BPH.Transforming growth factor-?(TGF-P)is an important cytokine that regulates EMT.Studies have found that RhoA/ROCK pathway may affect TGF-?-induced EMT response.But the role of RhoA/ROCK pathway and EMT in the pathogenesis of BPH is unclear.To investigate the role of RhoA/ROCK pathway in the development and progression of BPH,we investigated the expression and function of RhoA/ROCK pathway in BPH,and the role of RhoA/ROCK pathway in the EMT of prostate epithelial cells.Part ?:Expression and function of RhoA/Rho kinase pathway in human BPH tissuesObjective To investigate the expression and function of RhoA/ROCK pathway in human prostate tissue and explore whether RhoA/ROCK pathway was involved in the development of BPH,especially in the dynamic factors of BPH/LUTS.Methods Prostate tissues were collected from clinical surgery for the BPH and bladder cancer.Pathological staining and clinical symptom scores were used to distinguish BPH and normal groups.The expression of RhoA/ROCK,myosin light chain phosphatase(MLCP),myosin light chain(MLC)and protein phosphatase 1 regulatory inhibitor subunit 14A(CPI-17)were detected by PCR and western blotting.RhoA/ROCK was also detected by immunohistochemistry and immunofluorescence.To investigate the function in vitro,ROCK inhibitor Y-27632 was uesd to relax of the contraction of prostate tissue.Results 1)The mRNA expression of Rho A and ROCK1 in BPH group was significantly higher than that in normal group(P = 0.009),the expression of ROCK 1 increased to 1.8 times(P = 0.045)than normal group.2)Consistent with mRNA expression,the protein expression of RhoA and ROCK1 in BPH group was also increased,and the expression of CPI-17 and phosphorylated MLC were also increased in BPH group.3)The results of immunohistochemistry and immunofluorescence showed that RhoA and ROCK1 were both expressed in the epithelium and interstitial cells of prostate and were more pronounced in BPH tissues.4)10?M of Y-27632 could significantly relax prostate contraction induced by 10-5 M adrenaline in either BPH group or normal group.10?M of Y-27632-induced BPH tissue relaxed effect of was 38%,while in normal prostate tissue the relaxed efficacy was 17%,which indicated BPH group was more sensitive to Y-27632.Conclusions The expression of RhoA and ROCK1 increased in human BPH tissue,which leaded to increased expression of contraction-related downstream molecules.This might be involved in dynamic factors of BPH.And BPH tissue was more sensitive to ROCK inhibitor Y-27632.Part ?:RhoA/Rho kinase pathway is involved in TGF-?-induced prostate epithelial EMT processObjective The role of epithelial-mesenchymal transition(EMT)has been thoroughly studied in cancer invasion and metastasis.And the change of EMT markers in benign prostate hyperplasia(BPH)has been observed in present studies.However,the molecular mechanisms of this process in BPH is unclear.Methods The expression of E-cadherin,N-cadherin and vimentin were detected by immunohistochemical in BPH tissue.EMT in normal prostate epithelial cell line(RWPE-1)and hyperplastic prostate epithelial cell line(BPH-1)was induced by TGF-? treatment and the markers of EMT were detected by quantitative RT-PCR.And TGF-P induced expression of RhoA/ROCK pathway was detected.Then Y-27632,a specific inhibitor of ROCK was used together with TGF-P and the markers of EMT were detected again.Results 1)The expression of E-cadherin in prostatic epithelial cells from BPH tissue was lower than that in normal group,and the expression of vimentin and N-cadherin increased.2)EMT was observed in BPH-1 and RWPE-1 cell lines induced by TGF-?as the expression of epithelial marker E-cadherin decreased and the expression of mesenchymal markers vimentin and N-cadherin increased.3)In both cell lines,the mRNA expression of RhoA was up-regulated by TGF-?.After the co-applied with Y-27632,the TGF-? induced EMT was reversed.Y-27632 effectively reduced TGF-?induced mRNA expression of vimentin and N-cadherin.And the mRNA and protein expression of E-cadherin was increased.Conclusions This study suggested that TGF-P could induce EMT in BPH-1 and RWPE-1 cells,RhoA/Rock pathway might contribute to this process.