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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Dudas
Paul L
PL
Department of Immunology Research, Centocor Research and Development, Inc., 145 King of Prussia Road, R-4-2, Radnor, PA 19087, USA. pdudas@its.jnj.com
Argentieri
Rochelle L
RL
Farrell
Francis X
FX
D000199:Actins / Q000378:metabolism; D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000494:pharmacology*; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D000199:Actins; D055419:Bone Morphogenetic Protein 7; C526317:CCN2 protein, human; D015820:Cadherins; D015850:Interleukin-6; D016209:Interleukin-8; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D053773:Transforming Growth Factor beta1; D014746:Vimentin; D062826:Zonula Occludens-1 Protein; D055513:Connective Tissue Growth Factor