TLR4Signaling Augments TGF-β/EMT Responses In Prostatic Epithelial Cells And Its Associated Mechanism

BackgroundWith the improvement of living standard and the prolongation of average life-span, benign prostatic hyperplasia(BPH) becomes as a common and frequently-occurring disease among elderly men. The growth of the age and functional testicular is essential for the pathogenesis of BPH, but its pathogenesis is unclear. In recent years, the important function of the prostate gland inflammation in the pathogenesis of BPH has caused a lot of attention, some scholars put forward BPH is an immune-mediated inflammatory diseases.Toll like receptors4(TLR4) is the receptors of LPS, It plays a key role in congenital immune response to pathogenic microorganism. As the first line of congenital immune defense system, TLR4expressed in various immune cells, and expressed in various epithelial and endothelial cells. It is also reported that TLR4may participate in Epithelial-mesenchymal transition in prostatic epithelial cells, thus promote the occurrence and progress of prostate disease.Transforming growth factor beta1(TGF-betal) is a pleiotropic cytokine that involved in cell reconstruction process, such as cell proliferation, apoptosis, differentiation and migration. Bone morphogenetic protein and activation of transmembrane inhibitors (BAMBI), as the TGF-beta pseudo receptor, that can compete with TGF-beta type1receptor, to prevent type I and type II receptor and the ligand binding into functional complexes, which block TGF-beta signal transmission. BAMBI protein expression in the human body has been reported. However, the specific expression of BAMBI protein in human prostate tissue has rarely reported.Epithelial-mesenchymal transition refers to the Epithelial cells lose their polarity, and move freely among the cell matrix and interstitial cell phenotype transformation process. Study found that in BPH group, part of epithelial cells lose E-cadherin protein expression, at the same time get Vimentin protein expression, denoting the accumulation of BPH development process between stromal cells may be derived from the prostate epithelial cells. TGF-beta1epithelial cells is considered to be the key factor of the EMT, although the TGF-beta signaling pathway is relatively simple, but its regulating mechanism is very complicated. At present, the TGF-beta signaling pathways regulating and its relationship with other signaling pathways has become a new research hotspot.In order to explore the interaction mechanism of the LPS/TLR4, BAMBI and TGF-beta/Smad signaling pathways in the process of hyperplasia of prostate EMT signal transduction, we firstly apply immunohistochemistry and Immunofluorescence double dyeing technology to detect the expression and location of TLR4, BAMBI, p-Smad2/3in the tissue of BPH. Then the use of real-time quantitative PCR, Western blot and immunohistochemical technology to detect the index of BPH-1cells in EMT which stimulated by LPS and TGF-beta1, Finally we use adenovirus transfection technology build BAMBI high and low expression of BPH-1cell lines, TLR4signaling augments TGF-β/EMT responses in prostatic epithelial cells by downregulating BAMBI, has provided the new mentality for the pathogenesis of BPH.Method and Results1.The ralative expression of TLR4and p-Smad2/3in BPH tissues significantly raised:the expression of TLR4and p-Smad2/3in47cases of BPH tissues that combined with inflammatory was higher than that of unincorporated inflammation of15cases of BPH tissues, and there is no significant differencant expression was found in BAMBI(p<0.05).2.TLR4are negatively correlated with expression of BAMBI in inflammatory BPH tissues, while p-Smad2/3expression was positively related to it:We adopt Spearman analysis study the correlation of TLR4, BAMBI and p-Smad2/3expression in BPH tissues. The results shows that TLR4, BAMBI and p-Smad2/3has no direct correlation (r=-0.152,0.099, p=0.108,0.240), BAMBI and p-Smad2/3protein expression has little correlation (r=-0.298, p=0.041) in BPH without inflammation. However, in BPH with inflammation, TLR4are negatively correlated with BAMBI expression (r=-0.533, P=0.007), TLR4and p-Smad2/3expression were positively correlated (r=0.308, P=0.015), and BAMBI was negatively correlated with p-Smad2/3(r=-0.612, P=0.004).3. The co-expression of TLR4and BAMBI, TLR4and p-Smad2/3in BPH organizations:immunofluorescence shows that TLR4and BAMBI, TLR4and p-Smad2/3synchronously expressed in BPH tissues, especially in the BPH epithelial cells.4. LPS/TLR4induced BPH-1cells occur EMT by enhancing TGF-beta/Smad signaling pathways:We selected BPH-1cell line as the research object, immunohistochemical detection found that BPH-1cells express TLR4and BAMBI, and the expression of TLR4signiciantly down-regulated after stimulatied by LPS. BAMBI expression, on the other hand, is the opposite, and the expression of p-Smad2/3was negative. Real-time quantitative PCR, Western Blot and Immunohistochemistry are used to detect the expression of each group under EMT signaling pathways. The results showed that, E-cadherin mRNA and its protein expression in10ug/ml LPS+0.1ng/ml TGF-beta1group was significantly lower than that of blank control and negative control group (P<0.05), Vimentin and P-Smad2/3mRNA and protein expression was significantly higher than that of blank control and negative control group (P<0.05). After joining TLR4blockers CLI-095, the up-regulation of Vimentin and p-Smad2/3and the down-regulation of E-cadherin in10ug/ml LPS+0.1ng/ml TGF-beta1group have been inhibited(p<0.05).5. LPS/TLR4is significantly down-regalated the expression of BAMBI in BPH-1cells:Real-time quantitative PCR, Western Blot and Immunohistochemistry are used to detect the expression change of BAMBI proteins in BPH-1cells after stimulatied by LPS. The results showed that in LPS group the expression index of BAMBI mRNA and protein was significantly lower than that of the blank control group and LPS+CLI-095group(P<0.05).6. TLR4enhance TGF-beta/Smad-EMT signaling pathways by down-regulating BAMBI:With adenovirus transfection technique, we constructed the BAMBI protein with a low expression (AddnBambi) and a high expression (AdBambi) in BPH-1cell lines, and set the non-transfection BPH-1as a blank control. Using real-time quantitative PCR, Western Blot and immunohistochemical detection the expression of E-cadherin, Vimentin and P-Smad2/3between the3groups. Results show that, after the lOug/ml LPS+0.1ng/ml TGF-β1, the expression of p-Smad2/3and Vimentin in AdBambi group is significantly lower than that of the blank control group, E-cadherin expression is significantly higher than that of the control group (p<0.05), while no significant difference found in expression of p-Smad2/3, E cadherin and Vimentm and the blank control group in AddnBambi group(P>0.05).Conclusion 1. The expression of TLR4and p-Smad2/3is significantly up-regulated in BPH cells with inflammation, Which illustrates the TLR4and TGF-beta/Smad signaling pathway may be involved in the evelopment of BPH which promoted by inflammation.2. The expression of TLR4are negatively correlated with that of BAMBI in BPH cells with inflammation, while p-Smad2/3expression was positively related to it. The TLR4and BAMBI, TLR4and p-Smad2/3synchronously expressed in BPH tissues epithelial cells, prompting that TLR4and TGF-beta/Smad signaling pathways may communicate with BAMBI protein.3. BPH-1cells can express TLR4protein and BAMBI protein, but it can not express p-Smad2/3protein. And the expression of TLR4up-regalated after efected by LPS, whlie BAMBI expression down-regalated, illustrates the TLR4is significantly down-regalated the expression of BAMBI.4. LPS/TLR4enhance TGF-beta/Smad-EMT signaling pathways in BPH-1.5. With adenovirus transfection technique, we can build BPH-1cell lines with low or high BAMBI expression, overexpression of BAMBI can inhibit EMT in BPH-1. LPS/TLR4could enhance TGF-beta/Smad-EMT signaling pathways by down-regulating BAMBI in BPH-1.