Article

BMP-7 fails to attenuate TGF-beta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells.

Department of Immunology Research, Centocor Research and Development, Inc., 145 King of Prussia Road, R-4-2, Radnor, PA 19087, USA.
Nephrology Dialysis Transplantation (impact factor: 3.4). 01/2009; 24(5):1406-16. DOI:10.1093/ndt/gfn662
Source: PubMed

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.

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Keywords

alpha-SMA expression
 
alpha-smooth muscle actin
 
antagonizing TGF-beta1-stimulated epithelial-to-mesenchymal transition
 
attenuate EMT
 
BMP-7 inhibition
 
BMP-7 regulation
 
BMP-7-mediated reduction
 
e-cadherin expression
 
halt disease progression
 
IL-6 transcript expression
 
indicated EMT
 
NRK-49F rat renal fibroblasts
 
proximal tubule cells
 
rat renal fibroblasts
 
receptor expression
 
rodent models
 
TGF-beta1 expression
 
TGF-beta1 transcript expression
 
TGF-beta1-induced EMT
 
TGF-beta1-mediated e-cadherin downregulation