The role of Id proteins 1 and 2 in regulating phenotypic changes by TGFB1 and BMP7 in human renal epithelial cells

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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Smad Mediated Regulation of Inhibitor of DNA Binding 2 and Its Role in Phenotypic Maintenance of Human Renal Proximal Tubule Epithelial Cells

<div><p>The basic-Helix-Loop-Helix family (bHLH) of transcriptional factors plays a major role in regulating cellular proliferation, differentiation and phenotype maintenance. The downregulation of one of the members of bHLH family protein, inhibitor of DNA binding 2 (Id2) has been shown to induce de-differentiation of epithelial cells. Opposing regulators of epithelial/mesenchymal phenotype in renal proximal tubule epithelial cells (PTEC), TGFβ1 and BMP7 also have counter-regulatory effects in models of renal fibrosis. We investigated the regulation of Id2 by these growth factors in human PTECs and its implication in the expression of markers of epithelial versus myofibroblastic phenotype. Cellular Id2 levels were reduced by TGFβ1 treatment; this was prevented by co-incubation with BMP7. BMP7 alone increased cellular levels of Id2. TGFβ1 and BMP7 regulated Id2 through Smad2/3 and Smad1/5 dependent mechanisms respectively. TGFβ1 mediated Id2 suppression was essential for α-SMA induction in PTECs. Although Id2 over-expression prevented α-SMA induction, it did not prevent E-cadherin loss under the influence of TGFβ1. This suggests that the loss of gate keeper function of E-cadherin alone may not necessarily result in complete EMT and further transcriptional re-programming is essential to attain mesenchymal phenotype. Although BMP7 abolished TGFβ1 mediated α-SMA expression by restoring Id2 levels, the loss of Id2 was not sufficient to induce α-SMA expression even in the context of reduced E-cadherin expression. Hence, a reduction in Id2 is critical for TGFβ1-induced α-SMA expression in this model of human PTECs but is not sufficient in it self to induce α-SMA even in the context of reduced E-cadherin.</p> </div

Id2 was counter-regulated by TGFβ1 and BMP 7 in HKC 8 cells.

<p>HKC 8 cells were treated with either vehicle (0.1% BSA), TGFβ1 (5 ng/ml), BMP 7 (200 ng/ml) or both for 24 h and the expression of Id2 was determined by immunoblotting. The representative immunoblot shows the expression of Id2. TGFβ1 downregulated Id2 levels on the other hand BMP 7 upregulated Id2 levels and combined treatment of BMP 7 prevented TGFβ1 induced Id2 loss. The data is expressed as mean±SD (n = 3, ns = P>0.05, *<0.05).</p

BMP 7 inhibited TGFβ1 mediated α-SMA induction through Id2.

<p>HKC 8 cells were transfected with either Id2 siRNA or %GC content matched negative control siRNA for 24 h. After 24 h serum recovery and 24 h serum free period the cells were treated with either vehicle (0.1% BSA), TGFβ1 (5 ng/ml) or TGFβ1 (5 ng/ml) and BMP 7 (200 ng/ml) for 48 h. The expression of Id2 and α-SMA were determined by immunoblotting. The representative immunoblots shows the expression of Id2 and α-SMA (A). Id2 upregulation by BMP 7 was inhibited by siRNA (B). Id2 knock-down prevented BMP 7 inhibition of TGFβ1 mediated α-SMA induction (C). The data is expressed as mean±SD (n = 3, ns = P>0.05, *<0.05, **<0.01, ***<0.001).</p

BMP 7 induction of Id2 was prevented by combined Smad1/5 knock-down.

<p>HKC 8 cells were transfected with either Smad1 and Smad5 siRNAs or negative control siRNA for 24 h. After 24 h serum recovery and 24 h serum free period, the cells were treated with either vehicle (0.1% BSA) or BMP 7 (200 ng/ml) for a further 18 h. The cells were lysed and Id2, Smad1 and Smad5 expressions were assessed by immunoblotting. The representative immunoblots show the expression of Id2, Smad1 and Smad5. BMP 7 upregulation of Id2 was prevented by combined Smad1/5 knock-down. The data is expressed as mean±SD (n = 5, ns = P>0.05, ***<0.001).</p