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

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.


Introduction
Accumulation of extracellular matrix in chronic kidney disease is preceded by renal hypertrophy especially glomerular mesangial hypertrophy. Mesangial cell among the three cell types in the glomerulus acts as the predominant site for the synthesis of extracellular matrix proteins, which contribute to glomerular hypertrophy and renal fibrosis found in progressive chronic kidney diseases [1]. Various growth factors and cytokines produced by the infiltrating cells during the disease process and by the local kidney cells participate in the fibrotic process [2]. Among these, TGFb produced by the kidney cells and by the infiltrating macrophages plays a significant role in the pathogenesis of mesangial matrix expansion [3]. Increased glomerular expression of TGFb has been reported in both experimental and human kidney disease [3,4]. Mice with increased plasma TGFb1 levels displayed enhanced renal fibrosis [5]. On the other hand, blockage of TGFb1 prevented renal especially glomerular hypertrophy and fibrosis in mouse with diabetes [6,7].
TGFb initiates its signal transduction by binding to the type II receptor, which forms the oligomeric complex containing the type I receptor. In the tetrameric receptor complex, type II receptor phosphorylates type I receptor in the GS domain, which releases FKBP12 from the receptor, resulting in activation of the type I receptor serine threonine kinase. L45 loop of receptor kinase domain located immediately downstream of the GS segment interacts with the L3 loop of receptorspecific Smad 3 and 2 followed by phosphorylation of serine residues in the C-terminus of Smad protein [8,9]. This binding of the receptor to Smads is also facilitated by SARA, a FYVE domain containing protein, which immobilizes receptor-specific Smads to the plasma membrane [10]. Phosphorylated Smad dissociates from the receptor resulting in exposure of the nuclear import sequence and heterodimerization with the common Smad, Smad 4. The heteromeric Smad complex then translocates to the nucleus, recruits transcriptional co-activators or corepressors and regulates target gene expression [9,11,12]. Both in human and animal models of kidney fibrosis, TGFb-specific Smads are activated, which increases transcription of various collagens [13]. Deletion of Smad 3 in mice protects from fibrotic disorders of kidney [14,15,16]. Although both Smad 3 and Smad 2 act downstream of TGFb, unexpectedly, specific deletion of Smad 2 in kidney significantly enhanced Smad 3 activity, collagen matrix expansion and fibrosis, indicating that Smad 2 functions as a negative regulator of TGFb-driven renal fibrosis [17]. Along with this canonical signal transduction pathway, TGFb stimulates non-canonical signaling which includes activation of the tyrosine and serine threonine kinases, such as c-Src, Erk1/2, JNK and p38 MAP kinases [18,19,20]. Also, TGFb activates PI 3 kinase/Akt signaling [21,22]. More recently we and others have shown that TGFb regulates PI 3 kinase-dependent mTOR to increase cellular hypertrophy including mesangial cell hypertrophy [23,24,25]. miRNAs regulate expression of genes via post-transcriptional mechanism [26]. miRNAs are transcribed by RNA polymerase II similar to mRNAs and contain a 59 CAP and a 39 poly A tail [26,27,28]. In the nucleus primary transcripts of the miRNAs are processed by Drosha RNase III activity to produce stem-loop containing pre-miR, which are exported to the cytoplasm and further processed by Dicer in a complex containing TRBP. Recently, MCPIP1, an antagonist of Dicer, has been shown to act on the terminal loop of pre-miR to block Dicer activity [29]. Thus a concerted action of both these proteins produces , 22 nucleotide long double stranded RNAs. The miRNA guide strand is produced from this duplex, which then binds to Argonaute 2 in the RISC to interact with the specific miRNA recognition element present in the 39 UTR of target mRNAs [26,27]. This binding destabilizes the target mRNA and predominantly suppresses the translation of mRNA although degradation of mRNAs can also occur [26,27,28]. Conditional deletion of Dicer in the nephron progenitors showed that global loss of miRNAs induced a significant loss of nephron number [30,31]. Deletion of either Dicer or Drosha in the mouse glomerular podocytes showed loss of renal function with glomerulosclerosis, foot process effacement and proteinuria [31,32,33,34]. Interestingly, deletion of Dicer in proximal tubules protected mice from ischemia reperfusion injury [35]. The expression of myriad of miRNAs is altered in various kidney diseases [13,36,37,38]. Recently, a role of miR-21 in diabetes-induced pancreatic b cell death has been demonstrated [39]. Also, TGFb-mediated increase in miR-21 levels has been linked to the progression of disease in mouse models of fibrosis [40,41,42]. In the proximal tubular epithelial cells, TGFbstimulated matrix protein expression was linked to expression of miR-21 [41]. However, the signaling pathway miR-21 utilizes for fibrotic protein expression is poorly understood. Here we demonstrate that TGFb-stimulated expression of miR-21 in glomerular mesangial cells inhibits PTEN protein levels, which results in activation of Akt and mTORC1. Furthermore, we show that TGFb forces miR-21-targeted PTEN to upregulate protein synthesis and hypertrophy that is controlled by Akt/TORC1 signaling. Finally, we depict that miR-21-induced increase in two fibrotic matrix proteins fibronectin and collagen I (a2) uses PTEN/Akt/TORC1 pathway.

miR-21 Regulates PTEN-mediated Akt Activation in Human Glomerular Mesangial Cells
Recently, TGFb has been shown to enhance the expression of miR-21 in rodent glomerular mesangial cells in culture; however, the signaling role of miR-21 in TGFb-induced cellular hypertrophy and matrix protein expression has not been studied [40]. We confirmed the expression of mature miR-21 as well as pre-miR-21 in human mesangial cells (Fig. S1). The miR-21 target that mediates pathologic consequences of TGFb has not been identified in renal cells. We and others have recently identified the tumor suppressor protein PTEN as a regulator of TGFbinduced glomerular mesangial cell hypertrophy and matrix protein expression [25,43]. The 39UTR of PTEN mRNA has been experimentally validated as a target of miR-21 [44,45]. Therefore, to initiate studies involving miR-21 and PTEN, we tested the effect of TGFb on the reporter activity of a plasmid in which the firefly luciferase cDNA is fused to 39UTR of PTEN (PTEN 39UTR-Luc). Transient transfection assay using this plasmid in human mesangial cells showed significant repression of reporter activity in response to TGFb (Fig. 1A). Since miR-21 is increased in TGFb-stimulated mesangial cells (Fig. S1), we examined the effect of this miRNA. Plasmid-derived expression of miR-21 significantly inhibited the reporter activity of PTEN 39UTR-Luc (Fig. 1B, Fig.  S2A). Also, expression of miR-21 suppressed PTEN protein levels ( Fig. 1C and Fig. S2B) To confirm the role of miR-21, we used a plasmid vector called 'miR-21 Sponge', which contains 7 copies of bulged miR-21 binding site fused to the 39 end of GFP mRNA (Fig. S3A). Expression of this construct neutralizes miR-21 in cells [46]. Human mesangial cells were transiently transfected with PTEN 39UTR-Luc and miR-21 Sponge. Expression of miR-21 Sponge significantly increased the luciferase activity ( Fig. 1D). Concomitantly, miR-21 Sponge increased PTEN protein expression (Fig. 1E). Expression of GFP mRNA was used as a surrogate for miR-21 Sponge expression ( Fig. S3B and S3C). These results suggest that miR-21 targets PTEN in mesangial cells.
To elucidate the role of miR-21-in TGFb-mediated signal transduction, we used miR-21 Sponge. As expected TGFb inhibited the expression of PTEN protein in mesangial cells ( Fig. 2A) [25,43]. Expression of miR-21 Sponge significantly prevented TGFb-mediated decrease in PTEN levels ( Fig. 2A and Fig. S4A). PTEN is an endogenous inhibitor of PI 3 kinasedependent Akt activation [47,48]; it inhibits phosphorylation of Akt as we have previously shown in mesangial cells [25]. Thus, downregulation of PTEN in response to TGFb increased phosphorylation of Akt at both catalytic loop and hydrophobic motifs (Fig. 2B). Expression of miR-21 Sponge blocked TGFbstimulated phosphorylation of Akt ( Fig. 2B and Fig. S4B). Since both these phosphorylations of Akt regulate its enzymatic activity, we tested the phosphorylation of one of the endogenous substrates of this kinase, GSK3b as an index of Akt kinase activity. Concomitant with Akt phosphorylation, TGFb increased phosphorylation of GSK3b, which was prevented by expression of miR-21 Sponge (Fig. 2C and Fig. S4C).

miR-21 Regulates TGFb-stimulated Mesangial Cell Hypertrophy by PTEN/Akt/mTORC1 Signal Transduction
We and others have recently reported a role of PTEN in mesangial cell hypertrophy. Furthermore, TGFb promoted protein synthesis and mesangial cell hypertrophy [23,25,43]. We tested the involvement of miR-21-targeted PTEN in this process. As expected, TGFb increased protein synthesis due to reduced PTEN expression (Fig. 5A) [25]. Expression of miR-21 Sponge reversed TGFb-inhibited PTEN expression ( Fig   Sponge significantly inhibited TGFb-stimulated hypertrophy of mesangial cells (Fig. 5B and Fig. S7B). Downregulation of PTEN using siRNAs markedly prevented the inhibitory effect of miR-21 Sponge on TGFb-induced hypertrophy ( Fig. 5B and Fig. S7B). Next, we determined whether the miR-21-targeted PTEN uses Akt kinase for these processes. We used a plasmid vector containing Gag-Akt, which behaves as a constitutively active kinase [60]. Expression of Gag-Akt significantly reversed the inhibitory effect of miR-21 Sponge on TGFb-stimulated protein synthesis and hypertrophy ( Fig. 5C and 5D; Fig. S7C and S7D). These results suggest that TGFb-induced expression of miR-21 uses PTEN/Akt signaling in regulating mesangial cell protein synthesis necessary for hypertrophy.
The results described in Figure 4 demonstrate that miR-21 regulates mTORC1 activity, which phosphorylates 4EBP-1. mTORC1-mediated phosphorylation of 4EBP-1 results in its inactivation and initiation of protein synthesis, necessary for cellular hypertrophy including mesangial cell hypertrophy [23,55,57,59]. Therefore, we tested the role of mTORC1 in the action of miR-21 in TGFb-induced protein synthesis. We used a vector expressing a mutant mTOR that has constitutive mTORC1 activity [61,62]. Expression of the constitutively active (CA) mTOR along with miR-21 Sponge significantly reversed the suppressive effect of miR-21 Sponge on both TGFb-induced protein synthesis and hypertrophy of mesangial cells (Fig. 6A and 6B; Fig. S8A and S8B). Thus, our results demonstrate involvement of mTORC1 in the action of miR-21 in mesangial cell hypertrophy.

miR-21 uses PTEN/Akt/mTORC1 Signaling for TGFbinduced Matrix Protein Expression
TGFb contributes to renal fibrosis by stimulating the synthesis of matrix proteins such as fibronectin and collagen I (a2) [3]. TGFb stimulates expression of both these proteins in mesangial cells, which contribute to glomerulosclerosis [1]. We examined the role of miR-21-regulated PTEN in the expression of these proteins. As expected, incubation of mesangial cells with TGFb increased the expression of fibronectin and collagen I (a2) (Fig. 7). Expression of miR-21 Sponge blocked both fibronectin and collagen I(a2) expression in response to TGFb ( Fig. 7 and Fig. S9). To test if PTEN is involved in this inhibition by miR-21 Sponge, we used siRNAs against PTEN. Downregulation  of PTEN by siPTEN reversed the inhibition of both TGFbstimulated fibronectin and collagen expression by miR-21 Sponge (Fig. 7A and 7B and Fig. S9A and S9B). Since PTEN regulates activation of Akt kinase, we tested the involvement of Akt in miR-21 action. Expression of the constitutively active Gag-Akt prevented the miR-21 Sponge-induced suppression of TGFb-stimulated fibronectin and collagen I (a2) expression (Fig. 7C and 7D and Fig. S9C and  S9D). We have shown above that miR-21 controls TGFb-stimulated mTORC1 activity. We examined the involvement of this kinase in fibronectin and collagen expression. Expression of CA mTOR, which acts as constitutively active mTORC1 [62], reversed the miR-21 Sponge-mediated inhibition of both fibronectin and collagen expression in response to TGFb (Fig. 8A and 8B and Fig. S10A and S10B). Together these results indicate that miR-21-stimulated PTEN-Akt-mTORC1 signaling is required for TGFb-stimulated fibrotic protein expression in mesangial cells.

Discussion
We demonstrate that increased miR-21 uses the tumor suppressor protein PTEN as its downstream target to regulate Akt/mTORC1 signaling in response to TGFb in renal mesangial cells. We show that miR-21-targeted PTEN regulates TGFb-induced protein synthesis required for mesangial cell hypertrophy. miR-21-stimulated Akt/mTORC1 cascade forces expression of two fibrotic proteins, fibronectin and collagen, for induction of glomerulosclerosis (Fig. 9).
Recently we and others have shown a role of PTEN in TGFbforced diabetic renal glomerular hypertrophy in rats and mouse [25,43]. Downregulation of PTEN in renal glomeruli and in cultured mesangial cells in response to TGFb contributes to renal hypertrophy and matrix protein expression [25,43]. Abundance of PTEN is regulated at the levels of transcription, ubiquitination, phosphorylation, protein-protein interaction and oxidation [63]. More recently post-transcriptional control of expression of PTEN involving specific miRNAs has been elucidated. The miRNA, miR-192, regulates the expression of miR-216a and miR-217, both of which directly target the PTEN 39UTR [43]. Upregulation of these three miRNAs has been reported in renal glomeruli of diabetic mice and in mesangial cells incubated with high glucose or TGFb [43,64]. They induced mesangial cell hypertrophy and matrix protein collagen expression. In contrast to this observation, a recent study demonstrated decreased expression of miR-192 in mesangial and proximal tubular epithelial cells and in mouse diabetic kidney cortex [65]. This observation was confirmed in a human study where a microRNA expression profiling in renal   biopsies of patients with established diabetic nephropathy showed significantly reduced expression of miR-192, which correlated with low glomerular filtration rate and tubular fibrosis [66]. More recently, same investigators showed reduction in miR-192 in renal cells in response to TGFb1 [67]. Interestingly, Putta et al recently showed amelioration of renal fibrosis by administration of anti-miR-192 in a mouse model of diabetes, which involves TGFb action [68]. Several other miRNAs including miR-93, miR-29, miR-214, miR-377 and mir-200 have been shown to play important role in TGFb-driven renal fibrosis [13,36,40,69,70,71].
Role of the miRNA miR-21 in various diseases including fibrosis has been extensively studied. A recent study has shown increased expression of miR-21 in patients with acute kidney injury and chronic allograft dysfunction both of which exhibit fibrosis of the kidney [72]. Furthermore, positive and negative role of miR-21 has been reported in cardiomyocyte hypertrophy in vitro [73,74]. Moreover, tissue-specific action of miR-21 on PTEN expression is also reported. For example, miR-21 suppresses PTEN 39UTR in hepatic cancer cells. In contrast, it did not inhibit PTEN 39UTR in COS cells [45,75]. However, in renal mesangial cells we demonstrate suppression of PTEN 39UTR-driven reporter activity by miR-21, which regulates PTEN protein expression and its downstream Akt activation, resulting in phosphorylation of negative regulators of mTORC1 signaling (Figs. 1, 2, and 3). miR-21 is significantly upregulated in rodent models of diabetic nephropathy, glomerulonephritis, ischemia reperfusion injury (IRI) and ureteral obstruction (UUO) [40,41,42,72]. In IRI and UUO, increased expression of miR-21 was observed throughout the kidney especially in the glomerular cells. Less prominent expression was detected in the proximal epithelial compartment [72]. TGFb is involved in pathologies observed in these models in which increased expression of miR-21 was evident in proximal tubular epithelial and glomerular mesangial cells [40,41,42,72]. In concurrence, we found increased expression of miR-21 in human glomerular mesangial cells in response to TGFb (Fig. S1). Although many direct targets of miR-21 exist, the specific proteins, which may mediate the effect of miR-21 on kidney fibrosis, have not been identified. Recently, Chau et al identified PPARa, as a significant target that mediates the fibrotic disease process in the kidney [72]. In the present study, we demonstrate another target of miR-21 in TGFb-stimulated mesangial cells, PTEN, which regulates cellular hypertrophy and matrix protein expression.
We have previously reported that PI 3 kinase-activated Akt controls TGFb-stimulated hypertrophy and expression of plasminogen activator inhibitor-1, which contributes to the abundance of several matrix proteins in kidney tissues [23,82]. In mesangial cells TGFb-mediated expression of fibrotic proteins such as fibronectin and collagen is regulated by PI 3 kinase/Akt signal transduction and involves PTEN [22,25,83]. More recently Kato et al showed a role of PTEN in regulation of hypertrophy and, collagen and fibronectin by miR-216a [43]. In line with this observation, we now demonstrate a direct contribution of another miRNA, miR-21, which by targeting PTEN regulates mesangial cell hypertrophy and, expression of fibronectin and collagen in response to TGFb (Figs. 5, 7A and 7B). In fact our data support the notion that miR-21 regulates expression of both these fibrotic proteins by downregulation of PTEN to activate Akt kinase ( Fig. 7C and 7D).
The results from our laboratory and other have established a role of mTORC1 in cellular hypertrophy especially in renal cell hypertrophy [23,24,52,84,85]. Activation of mTORC1 involving miRNAs has been reported. Along with PRAS40 regulation of raptor, AMP-activated protein kinase (AMPK) phosphorylates raptor, resulting in inhibition of mTORC1 [86]. Binding of AMPK-phosphorylated raptor to 14-3-3f is necessary for inhibition of mTORC1 activity [86]. miR-451 directly targets 14-3-3f to induce unrestrained mTORC1 activity [87]. Additionally, miR-451 reduces the levels of AMPK-activating LKB1 kinase cofactor CAB39 (calcium binding protein 39) by binding to the 39UTR of its mRNA [88,89]. Similarly, miR-17-92 cluster-coded miR-19 directly targets the AMPK a1 catalytic subunit to inhibit its activity, thus promotes mTORC1 activation [90]. More recently miR-221 has been shown to downregulate REDD1, which activates tuberin by dissociating it from 14-3-3 and thus inhibiting mTORC1 activity [91,92]. In many cancer cells, downregulation of miR-100, miR-101 and miR-199-3p, which bind to the 39UTR of mTOR mRNA to block its protein levels, has been shown  [93,94,95]. However, the role of these miRNAs in kidney cells and in the setting of renal fibrosis has not been investigated. We present data showing involvement of the miRNA miR-21 in activation of mTORC1 in response to the fibrotic cytokine TGFb (Fig. 4). Our results demonstrate that TGFb-stimulated miR-21 regulates hypertrophy of mesangial cells by targeting PTEN via activation of mTORC1 (Fig. 6). Furthermore, our results demonstrate a role of miR-21 targeted mTORC1 in TGFbstimulated expression of both fibronectin and collagen (Fig. 8).
Although administration of anti-miR-21 has been shown to block fibrosis in renal tissues, whether it derepresses any specific target protein has not been examined [41,72]. Confirmation of derepression of target proteins such as PTEN in the miR-21treated animal models of fibrosis will be necessary to establish the specificity of the therapy. Furthermore, it is important to establish a reciprocal correlation between expression of miR-21 and PTEN in renal tissues of patients with renal fibrosis. It will be beneficial if this correlation can be detected in subjects before the onset of the disease such as in prediabetics.

Cell Culture and Transfection
Preparation of human renal glomerular mesangial cells were described previously [97]. Frozen cells were thawed and grown in DMEM with 10% fetal bovine serum essentially as described previously [98,99,100]. The cells were used between passages 8 and 12. The cells were transfected with indicated plasmids in 12well or 24 well culture plates using Fugene HD transfection reagent as described previously [23,55,62,82].

Cell Lysis and Immunoblotting
For each experiment, cells were washed 2x with PBS and radioimmunoprecipitation buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM Na 3 VO 4 , 1 mM PMSF and 0.1% protease inhibitor cocktail) was added. The cell monolayer was incubated at 4uC for 30 minutes. The monolayer was scraped and centrifuged for 20 minutes at 4uC. The supernatant was collected and protein was estimated. Equal amounts of cell lysates were separated by SDS polyacrylamide gel electrophoresis. The separated proteins were transferred to PVDF membrane. Immunoblotting was carried out using indicated antibodies. The protein bands were developed using HRPconjugated secondary antibodies with ECL chemiluminiscent reagent as described previously [23,55,62,82].

Real Time Quantitative RT-PCR
Total RNA was prepared using TRIZol reagent as described [101]. First strand cDNA was synthesized by RT 2 kit according to the instruction provided by the vendor. qRT-PCR was performed in real-time PCR machine (7500, Applied Biosystems). U6 primers were used for normalization. Each sample was analyzed in duplicate. PCR conditions were as follows: 94uC for 10 minutes, followed by 40 cycles at 94uC for 30 seconds, 56uC for 30 seconds, 72uC for 30 seconds. Primers used for detection of pre-miR-21 were: Forward, 59-TGTCGGGTAGCTTATCAGAC-39; Reverse, 59-TTCAGACAGCCCATCGACTG-39.

End Point RT-PCR
To determine the expression of miR-21 Sponge, we detected the levels of GFP mRNA as a surrogate [46]. One mg of total RNA from miR-21 Sponge-transected mesangial cells was reverse transcribed and amplified to detect GFP mRNA. PCR conditions were: 94uC for 10 minutes, followed by 40 cycles at 94uC for 30 seconds, 58uC for 30 seconds, 72uC for 30 seconds. The primers used for detection of GFP mRNA are as follows: Forward primer: 59-ACGGCAAGCTGACCCTGAAG-39; Reverse primer: 59-GGGTGCTCAGGTAGTGGTTG-39.

Luciferase Activity
Lysates from reporter-transfected mesangial cells were used to measure luciferase activity using a kit as described previously [62,82,102]. The data are presented as mean of luciferase activity per microgram protein as arbitrary units 6 SE of indicated measurements as described in the figure legends.

Protein Synthesis and Hypertrophy
Transfected mesangial cells were serum-starved 16 hours followed by incubation with 2 ng/ml TGFb for 24 hours. Protein synthesis was determined as 35 S-methionine incorporation as described previously [25,55]. For measurement of hypertrophy, after incubation, the cells were trypsinized and counted in the hemocytometer. The cells were then centrifuged at 4000xg for 5 minutes and the cell pellets were washed with PBS, lysed in RIPA buffer and protein content was measured. Hypertrophy was determined as increase in protein content per cell as described previously [23,25,55].

Statistics
The data were analyzed by paired t-test. Where necessary the significance of the data was determined by ANOVA followed by Student-Newman-Keuls analysis as described previously [23,25,55,62]. p value less than 0.05 was considered as significant. Figure S1 Expression of miR-21 in response to TGFb in human mesangial cells. Serum-starved mesangial cells were incubated with 2 ng/ml TGFb for 24 hours. Total RNA from these cells were used for detection of Pre-miR-21 (panel A) and mature miR-21 (panel B) as described in the Materials and Methods. For panel A, mean 6 SE of triplicate measurements is shown. *p = 0.0005 vs control. For panel B, mean 6 SE of six measurements is shown. *p = 0.0001 vs control. (TIF) Figure S2 Expression of CMV miR-21 for the results described in Figure 1B and 1C. Mesangial cells were transfected with CMV miR-21 as described in the legend of Figure 1B and 1C. Total RNAs were used to detect mature miR-21 levels by qRT-PCR as described in the Materials and Methods. (TIF) Figure S3 Expression of miR-21 Sponge for the results described in Figure 1D Figure 6A and 6B. Mesangial cells were transfected with miR-21 Sponge and CA mTOR and treated with TGFb as described in the legend of Figure 6A Figure 8A and 8B. Mesangial cells were transfected with miR-21 Sponge and CA mTOR and treated with TGFb as described in the legend of Figure 8A and 8B. Total RNAs were used to detect GFP and GAPDH as indicated.