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Title: The role of Id proteins 1 and 2 in regulating phenotypic changes by TGFB1 and BMP7 in human renal epithelial cells
Author: Veerasamy, Mangalakumar
Awarding Body: University of London
Current Institution: University of London
Date of Award: 2012
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Tubulo-interstitial fibrosis (TIF) is a key feature of chronic kidney diseases (CKD). Epithelial mesenchymal transition of PTECs is considered to contribute to the interstitial fibroblast pool which secretes excessive matrix proteins seen in TIF. TGFpl plays a crucial role in TIF including mediating EMT. In animal models of CKD treatment with bone morphogenic protein 7 (BMP 7), a member of TGF superfamily improved the histology and renal function. However, the cellular signalling mechanisms involved in the anti- fibrotic effects of BMP 7, in particular whether BMP 7 inhibits TGFp 1 mediated EMT of human PTECs have not been studied. Experiments were performed in HKC 8 cells; a virally transformed human PTEC model. The expression of cell surface receptors for TGF superfamily and phosphorylation of TGFp 1 and BMP 7 Smads were studied by immunoblotting, and the nuclear translocation of Smad proteins was studied by immunofluorescence. The expressions of E-cadherin and (l-SMA were studied with TGFp 1 and BMP 7 treatment individually and in combination. siRNAs were used to knock-down Smadl and 5 proteins, to identify their role in BMP 7 regulation of markers of EMT. TGFp 1 and BMP 7 regulation of Id2 expression was studied at the protein level and the Smad signalling regulating this process was studied by '. silencing their expression with siRNA. siRNAs were used to study the role of Id2 on the expression of E-cadherin and (l-SMA with TGFp 1 and BMP 7 stimulation. Plasmid vector expressing Id2 was used to overexpress Id2 to study its role in TGFp 1 regulation of , markers ofEMT. Idl expression was studied at the protein level with BMP 7, and the Smad signalling involved in this process was studied by silencing their expression with siRNAs. The role of Id 1 in BMP 7 regulation of E-cadherin and (l-SMA was studied by silencing its expression by siRNA. The interaction of Idl and Id2 with E2A gene products was studied by nuclear co-immunoprecipitation. HKC 8 cells express TGF type 11, Alkl, Alk2, Alk3, Alk5 and Alk6 receptors. TGFpl and BMP 7 activated their respective receptor Smads concurrently during combined treatment. The nuclear translocation of their respective receptor Smads was not inhibited by the other during combined treatment. BMP 7 do~egulated E-cadherin, and it had an additive effect with TGFp 1 in this process and this was a Smad 1/5 dependent event. BMP 7 inhibited TGFp 1 induction of (l-SMA through Smad 1/5 signalling. TGFp 1 downregulated Id2 through Smad2/3 signalling and BMP 7 counter-regulated this through Smad1/5 signalling. IV Id2 gene silencing prevented BMP 7 inhibition of TGF~ I mediated a-SMA' expression. Id2 overexpression prevented TGF~ I mediated a-SMA expression. BMP 7 increased expression of Idl through non-Smadl/5 pathway and Id l gene silencing resulted in induction of a-SMA with BMP 7 stimulation. Both Idl and Id2 were co- immunoprecipitated with EI2 and E47 in the nuclear extract. In conclusion, these studies show that the anti-fibrotic effect of BMP 7 is mediated by inhibition of TGF~ 1 mediated induction of a-SMA and the myofibroblastic transition of PTECs in this in vitro model. The activation of Smadl/5 and Id2 signalling is involved in this counter-regulation. Although BMP 7 itself downregulated E-cadherin through Smadl/5 signalling, the concurrent induction of Idl through non-Smadl/5 pathway prevented de novo induction of a-SMA and myofibroblastic transition of PTECs. Id I and Id2 were shown to bind with E2A gene products, but their role in the expressions of markers of EMT in this model needs further confirmation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available