Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning

Transcriptome analysis of BMP2+ cells in comparison to the undifferentiated BMP2 ES cells and the control population from 7-day old embryoid bodies led to the identification of 479 specifically upregulated and 193 downregulated transcripts

TGFβ-Stimulated MicroRNA-21 Utilizes PTEN to Orchestrate AKT/mTORC1 Signaling for Mesangial Cell Hypertrophy and Matrix Expansion

<div><p>Transforming growth factor-β (TGFβ) promotes glomerular hypertrophy and matrix expansion, leading to glomerulosclerosis. MicroRNAs are well suited to promote fibrosis because they can repress gene expression, which negatively regulate the fibrotic process. Recent cellular and animal studies have revealed enhanced expression of microRNA, miR-21, in renal cells in response to TGFβ. Specific miR-21 targets downstream of TGFβ receptor activation that control cell hypertrophy and matrix protein expression have not been studied. Using 3′UTR-driven luciferase reporter, we identified the tumor suppressor protein PTEN as a target of TGFβ-stimulated miR-21 in glomerular mesangial cells. Expression of miR-21 Sponge, which quenches endogenous miR-21 levels, reversed TGFβ-induced suppression of PTEN. Additionally, miR-21 Sponge inhibited TGFβ-stimulated phosphorylation of Akt kinase, resulting in attenuation of phosphorylation of its substrate GSK3β. Tuberin and PRAS40, two other Akt substrates, and endogenous inhibitors of mTORC1, regulate mesangial cell hypertrophy. Neutralization of endogenous miR-21 abrogated TGFβ-stimulated phosphorylation of tuberin and PRAS40, leading to inhibition of phosphorylation of S6 kinase, mTOR and 4EBP-1. Moreover, downregulation of miR-21 significantly suppressed TGFβ-induced protein synthesis and hypertrophy, which were reversed by siRNA-targeted inhibition of PTEN expression. Similarly, expression of constitutively active Akt kinase reversed the miR-21 Sponge-mediated inhibition of TGFβ-induced protein synthesis and hypertrophy. Furthermore, expression of constitutively active mTORC1 prevented the miR-21 Sponge-induced suppression of mesangial cell protein synthesis and hypertrophy by TGFβ. Finally, we show that miR-21 Sponge inhibited TGFβ-stimulated fibronectin and collagen expression. Suppression of PTEN expression and expression of both constitutively active Akt kinase and mTORC1 independently reversed this miR-21-mediated inhibition of TGFβ-induced fibronectin and collagen expression. Our results uncover an essential role of TGFβ-induced expression of miR-21, which targets PTEN to initiate a non-canonical signaling circuit involving Akt/mTORC1 axis for mesangial cell hypertrophy and matrix protein synthesis.</p> </div

miR-21/PTEN/Akt axis regulates mesangial cell matrix protein expression in response to TGFβ.

<p>Mesangial cells were transfected with miR-21 Sponge and siPTEN or scrambled RNA as indicated in panels A and B. Similarly, mesangial cells were transfected with miR-21 Sponge and Gag-Akt as indicated in panels C and D. The transfected cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with fibronectin, PTEN, actin (panel A), collagen I (α2), PTEN, actin (panel B), fibronectin, Akt, actin (panel C) and collagen I (α2), Akt, actin (panel D) antibodies as indicated.</p

Akt2 causes TGFβ-induced deptor downregulation facilitating mTOR to drive podocyte hypertrophy and matrix protein expression.

TGFβ promotes podocyte hypertrophy and expression of matrix proteins in fibrotic kidney diseases such as diabetic nephropathy. Both mTORC1 and mTORC2 are hyperactive in response to TGFβ in various renal diseases. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. We identified that deptor downregulation by TGFβ maintains hyperactive mTOR in podocytes. To unravel the mechanism, we found that TGFβ -initiated noncanonical signaling controls deptor inhibition. Pharmacological inhibitor of PI 3 kinase, Ly 294002 and pan Akt kinase inhibitor MK 2206 prevented the TGFβ induced downregulation of deptor, resulting in suppression of both mTORC1 and mTORC2 activities. However, specific isoform of Akt involved in this process is not known. We identified Akt2 as predominant isoform expressed in kidney cortex, glomeruli and podocytes. TGFβ time-dependently increased the activating phosphorylation of Akt2. Expression of dominant negative PI 3 kinase and its signaling inhibitor PTEN blocked Akt2 phosphorylation by TGFβ. Inhibition of Akt2 using a phospho-deficient mutant that inactivates its kinase activity, as well as siRNA against the kinase markedly diminished TGFβ -mediated deptor suppression, its association with mTOR and activation of mTORC1 and mTORC2. Importantly, inhibition of Akt2 blocked TGFβ -induced podocyte hypertrophy and expression of the matrix protein fibronectin. This inhibition was reversed by the downregulation of deptor. Interestingly, we detected increased phosphorylation of Akt2 concomitant with TGFβ expression in the kidneys of diabetic rats. Thus, our data identify previously unrecognized Akt2 kinase as a driver of TGFβ induced deptor downregulation and sustained mTORC1 and mTORC2 activation. Furthermore, we provide the first evidence that deptor downstream of Akt2 contributes to podocyte hypertrophy and matrix protein expression found in glomerulosclerosis in different renal diseases

miR-21/PTEN/Akt axis regulates mesangial cell protein synthesis and hypertrophy in response to TGFβ.

<p>Mesangial cells were cotransfected with miR-21 Sponge and siRNAs targeting PTEN mRNA (siPTEN) or scrambled RNA (Scr) (panels A and B). Mesangial cells were cotransfected with miR-21 Sponge and constitutively active Gag-Akt as indicated (panels C and D). The transfected cells were starved for 16 hours prior to incubation with 2 ng/ml TGFβ for 24 hours. Protein synthesis (panels A and C) and hypertrophy (panels B and D) were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#s4" target="_blank">Materials and Methods </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Mahimainathan1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Dey1" target="_blank">[55]</a>. Mean ± SE of 3 measurements is shown. For panel A, *p<0.01 vs control; **p<0.01 vs TGFβ; #p<0.05 vs miR-21 Sponge plus TGFβ. For panel B, *p<0.05 vs control; **p<0.05 vs TGFβ; #p<0.05 vs miR-21 Sponge plus TGFβ. For panel C, *p<0.05 vs control; **p<0.05 vs TGFβ; #p<0.05 vs miR-21 Sponge plus TGFβ. For panel D, *p<0.01 vs control; **p<0.05 vs TGFβ; #p<0.05 vs miR-21 Sponge plus TGFβ. Bottom panels show expression of PTEN and Akt in representative samples. Actin expression was used as a control for immunoblotting.</p

miR-21 targets PTEN to regulate activation of Akt kinase in response to TGFβ.

<p>Mesangial cells were transfected with miR-21 Sponge or vector followed by incubation with 2 ng/ml TGFβ for 24 hours. Cell lysates were immunoblotted with PTEN, actin (panel A), phospho-Akt (Ser-473), phospho-Akt (Thr-308), Akt (panel B), phospho-GSK3β and GSK3β (panel C) antibodies as indicated.</p

miR-21 Sponge inhibits TGFβ-stimulated phosphorylations of tuberin and PRAS40.

<p>Glomerular mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-tuberin (Thr-1462), tuberin (panel A), phospho-PRAS40 (Thr-246) and PRAS40 (panel B) antibodies as indicated.</p

Constitutively active mTORC1 prevents the inhibition of miR-21 Sponge on TGFβ-stimulated fibronectin and collagen expression.

<p>Glomerular mesangial cells were transfected with miR-21 Sponge and CA mTOR plasmids as indicated. The cells were incubated with TGFβ for 24 hours. The cell lysates were immunoblotted with fibronectin (panel A) and collagen I (α2) (panel B) antibodies. Immunoblots of mTOR and actin are shown at the bottom.</p

TGFβ-stimulated miR-21 targets PTEN 3′UTR to inhibit PTEN expression.

<p>(A) Human glomerular mesangial cells were transfected with PTEN 3′UTR-containing luciferase (PTEN 3′UTR-Luc) reporter plasmid PTEN 3′UTR-Luc. Transfected cells were serum-starved for 16 hours followed by incubation with 2 ng/ml TGFβ for 24 hours. The cell lysates were assayed for luciferase activity as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#s4" target="_blank">Materials and Methods </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das2" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das3" target="_blank">[82]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Mahimainathan2" target="_blank">[102]</a>. Mean ± SE of six measurements is shown. *p = 0.018 vs control. (B and C) Mesangial cells were cotransfected with PTEN 3′UTR-Luc and CMV-miR-21 (expressing mature miR-21). The cell lysates were assayed for luciferase activity as described (panel B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das2" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das3" target="_blank">[82]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Mahimainathan2" target="_blank">[102]</a>. Mean ± SE of triplicate measurements is shown; *p = 0.003 vs vector. For panel C, the cell lysates were immunoblotted with PTEN and actin antibodies. (D and E) Mesangial cells were transfected with PTEN 3′UTR-Luc plus miR-21 Sponge. For panel D the cell lysates were assayed for luciferase activity as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das2" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Das3" target="_blank">[82]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042316#pone.0042316-Mahimainathan2" target="_blank">[102]</a>. Mean ± SE of 12 measurements is shown; *p = 0.0001 vs vector. For panel E, the cell lysates were immunoblotted with PTEN and actin antibodies.</p

miR-21 Sponge blocks mTORC1 activity in response to TGFβ.

<p>Mesangial cells were transfected with miR-21 Sponge or vector. The serum-starved cells were incubated with 2 ng/ml TGFβ for 24 hours. The cell lysates were immunoblotted with phospho-S6 kinase (Thr-389), S6 kinase (panel A), phospho-mTOR (Ser-2448), mTOR (panel B), phospho-4EBP-1 (Thr-34/46), phospho-4EBP-1 (Ser-65) and 4EBP-1 (panel C) antibodies as indicated.</p