BMP-7 fails to attenuate TGF-beta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells.
ABSTRACT In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-beta1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells.
EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-beta1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta1 treatment in RPTEC and HK-2 cells.
In RPTEC and HK-2 cells, TGF-beta1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta1 expression. TGF-beta1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta1 with an anti-TGF-beta1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 microg/ml) with TGF-beta1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-beta1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-beta1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation of BMPR-IA, BMP-7-mediated reduction of IL-6 transcript expression and BMP-7-mediated reduction of secreted IL-6 and IL-8 proteins.
In the present study, despite confirming BMP-7 regulation of receptor expression and induction of downstream signalling events, we were unable to demonstrate BMP-7 inhibition of EMT in either primary or immortalized human proximal tubule cells. Moreover, we were unable to demonstrate BMP-7-stimulated MET in rat renal fibroblasts. A protective effect was however observed at an elevated BMP-7 concentration in mouse renal tubular epithelial cells.
Article: Bone morphogenetic protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis.[show abstract] [hide abstract]
ABSTRACT: Bone Morphogenetic Protein (BMP)-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT)-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8) expression was quantified in proximal tubular cells (HK-2). Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-β induced epithelial-to-mesenchymal transition (EMT), expression of TGF-β receptor type I (TGF-βRI) and phosphorylated Smad 2 (pSmad 2) as well as on TNF-α induced apoptosis of proximal tubular cells. BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml) were able to reverse TNF-α-induced apoptosis and TGF-β-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-βRI. In addition, BMP-7 was able to reverse TGF-β-induced phosphorylation of Smad 2. The findings suggest a protective role for BMP-7 by counteracting the TGF-β and TNF-α-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease.BMC Nephrology 11/2010; 11:31. · 2.18 Impact Factor
Article: The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma.[show abstract] [hide abstract]
ABSTRACT: Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown. We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells. An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.PLoS ONE 01/2011; 6(8):e23057. · 4.09 Impact Factor
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ABSTRACT: TGF-β1 has been long considered as a key mediator in renal fibrosis and induces renal scarring largely by activating its downstream Smad signaling pathway. Interestingly, while mice overexpressing active TGF-β1 develop progressive renal injury, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Under disease conditions, Smad2 and Smad3 are highly activated, while Smad7 is degraded through the ubiquitin proteasome degradation mechanism. In addition to TGF-β1, many pathogenic mediators such as angiotensin II and advanced glycation end products can also activate the Smad pathway via both TGF-β-dependent and independent mechanisms. Smads interact with other signaling pathways, such as the MAPK and NF-κB pathways, to positively or negatively regulate renal inflammation and fibrosis. Studies from gene knockout mice demonstrate that TGF-β1 acts by stimulating its downstream Smads to diversely regulate kidney injury. In the context of renal fibrosis and inflammation, Smad3 is pathogenic, while Smad2 and Smad7 are protective. Smad4 exerts its diverse roles by transcriptionally enhancing Smad3-mediated renal fibrosis while inhibiting NF-κB-driven renal inflammation via a Smad7-dependent mechanism. Furthermore, we also demonstrated that TGF-β1 acts by stimulating Smad3 to positively or negatively regulate microRNAs to exert its fibrotic role in kidney disease. In conclusion, TGF-β/Smad signaling is a major pathway leading to kidney disease. Smad3 is a key mediator in renal fibrosis and inflammation, whereas Smad2 and Smad7 are renoprotective. Smad4 exerts its diverse role in promoting renal fibrosis while inhibiting inflammation. Thus, targeting the downstream TGF-β/Smad3 signaling pathway by gene transfer of either Smad7 or Smad3-dependent microRNAs may represent a specific and effective therapeutic strategy for kidney disease.International journal of biological sciences 01/2011; 7(7):1056-67. · 2.70 Impact Factor