Posttranscriptional upregulation of Tsc-22 by Ybx1, a target of miR-216a, mediates TGF-β-induced collagen expression in kidney cells

Mitsuo Kato1, Lin Wang2, Sumanth Putta1,2, Mei Wang1, Hang Yuan1, 4, Guangdong Sun1,4, Linda Lanting1, Ivan Todorov1,2, John J. Rossi2,3 and Rama Natarajan1,2

1Gonda Diabetes Center, 2 Irell & Manella Graduate School of Biological Sciences, 3Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 1500 East Duarte Road,

Duarte, CA 91010, 4Division of Nephrology, 2nd Hospital of Jilin University, Changchun, China 130041 Running head: miR-216a, Ybx1 and Tsc-22 in collagen regulation

Address Correspondence to: Rama Natarajan, Ph. D. 1500 East Duarte Road, Duarte, CA 91010. Fax: 626-301-8136; Email; RNatarajan@coh.org

Increased accumulation of

extracellular matrix proteins and hypertrophy induced by Transforming growth factor-beta1 (TGF-β) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy (DN). Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-β increased microRNA-216a (miR-216a) levels in mouse MC, with parallel downregulation of Ybx1, a miR-216a target and RNA binding protein. TGF-β also enhanced protein levels of Tsc-22 (TGF-β stimulated clone 22) and collagen type I alpha-2 (Col1a2) expression in MC through far-upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies (P-bodies) in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-β, miR-216a mimics or Ybx1 shRNA to increase Tsc-22 protein levels, but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-β could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-IP assays revealed that TGF-β promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-β-induced Col1a2 in MC related to the pathogenesis of DN.

Diabetic Nephropathy (DN) is one of the major complications of diabetes. The main

features of DN include renal glomerular and tubular hypertrophy and fibrosis due to progressive accumulation of extracellular matrix (ECM) proteins in component kidney cells (1). Transforming growth factor-beta1 (TGF-β) levels are increased in renal cells including mesangial (MC) and other cells under diabetic conditions and upregulate ECM proteins such as collagens. TGF-β promotes ECM gene expression via both Smad transcription factors and E-box dependent mechanisms (2,3). E-box elements are located in the far-upstream enhancer region of the collagen gene (4-6). E-box repressors, Zeb1 (also known as δEF1) and Zeb2 (also known as Smad-interacting protein1, SIP1), repress target genes by binding to E-boxes in their promoters (7,8). They are also repressors of collagen type 1 and type 2 genes (4,5). TGF-β increases Col1a2 expression in MC by downregulating Zeb1 and Zeb2 (9). E-boxes are positively regulated by transcription factor E3 (TFE3)(3,10-12) as well as by Upstream Stimulatory Factors (USFs) (13-17). TGF-β is also reported to promote MC hypertrophy by activating phosphatidylinositol 3-kinase (PI3K)/Akt kinase pathway (10,18-20), as well as other cell types (21,22). PI3K phosphorylates membrane phosphatidylinositol-diphosphate (PIP2) to generate the second messenger lipid PIP3, which in turn activates Akt. Activated Akt phosphorylates several downstream proteins to control cell survival and protein synthesis. PTEN (phosphatase and tensin homologue), a tumor suppressor that is frequently mutated in various human cancers, inhibits Akt activation by dephosphorylating PIP3 (23). Decreased Pten expression levels and enhanced Akt activation are associated with hypertrophy in MC treated with high glucose or TGF-β (10,19).

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http://www.jbc.org/cgi/doi/10.1074/jbc.M110.165027The latest version is at JBC Papers in Press. Published on August 16, 2010 as Manuscript M110.165027

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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PI3K/Akt signal transduction is also involved in ECM gene upregulation (20,24).

MicroRNAs (miRNAs) are short non-coding RNAs of ~22 nucleotides that induce post-transcriptional gene repression by blocking protein translation (by binding to the 3’UTR of their target genes) or by inducing mRNA degradation (25,26). miRNAs also repress gene expression by recruiting target mRNAs to cytoplasmic RNA processing bodies (P-bodies) for RNA storage, decapping, deadenylation or degradation (27). At least 60% of human protein-coding genes expressed in the genome are downregulated by miRNAs (25). Recent evidence suggests that miRNAs regulate the expression of key genes relevant to cancer and kidney diseases (28-30). miR-192 is upregulated in the renal glomeruli of diabetic mice and in glomerular MC treated with TGF-β, and induces Col1a2 gene by inhibiting Zeb1 and Zeb2 (E-box repressors) (9). miR-192 is also upregulated in renal fibrosis (mouse kidneys after unilateral ureteral obstruction and a rat model of remnant kidney disease) and tubular epithelial cells treated with TGF-β in the Smad3-dependent manner (31). On the other hand, miR-192 is downregulated by TGF-β and inhibits E-Cadherin expression through upregulation of Zeb1 and Zeb2 in epithelial-to-mesenchymal transition (32,33). miR-377 increases fibronectin in human MCs by targeting PAK1 and MnSOD (34). Vascular endothelial growth factor, a target of miR-93, is upregulated by hyperglycemic conditions through downregulation of miR-93, and collagen and fibronectin are upregulated in parallel in kidney cells (35). A miRNA cluster (miR-216a and miR-217) targeting PTEN mediates TGF-β-induced activation of Akt in mouse MC (10). miR-216a and miR-217 are both located in an intron of a non-coding RNA, RP23-298H6.1-001 (RP23). Interestingly, this non-coding RNA (as well as miR-216a and miR-217) is regulated by TGF-β and miR-192 through E-boxes present in the RP23 promoter region. These TGF-β-triggered miRNA circuits and downstream signaling cascades result in enhanced ECM accumulation, hypertrophy, cell survival and oxidant stress in MC related to the pathogenesis of DN (10).

Several mechanisms of enhanced mRNA translation of ECM and other proteins

related to diabetic kidney diseases have been proposed (1,36). A recent report showed that hnRNP E1 phosphorylation by Akt activated by TGF-β upregulates Dab2 and ILEI translation (37). However, a potential role for miRNA-mediated regulation of mRNA translation in response to TGF-β in renal cells has not been fully studied. Here we show that TGF-β induces miR-216a in MC and downregulates its target Ybx1 which regulates the translation of a binding partner RNA, Tsc-22. Furthermore, we uncovered a new mechanism by which Ybx1, Tsc-22 and Tfe3 coordinately mediate TGF-β-induced Col1a2 expression in MC.

EXPERIMENTAL PROCEDURES

Cell culture- Primary mouse MC (MMC) were isolated and cultured as described (9). Recombinant human TGF-β1 was from R&D Systems, Inc. Mouse kidney cell line TCMK-1 was maintained as reported (9). Mouse models- All animal studies were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee. Induction of diabetes by STZ injections in C57BL/6 mice was carried out as described (9,10). Mice were used 7 weeks after the onset of diabetes. db/db mice and genetic control db/+ mice (10 weeks old) were from Jackson Laboratories (Bar Harbor, Maine). Glomeruli were sieved from the kidney as described (9). Real time quantitative PCR (qPCR)- Real-time qPCRs were performed using SYBR Green PCR Master Mix and 7300 Realtime PCR System (Applied Biosystems) as reported (9). Real time qPCR was also used to detect miR-216a, as reported (9,10). miRNA expression levels were also confirmed by the miScript System, Qiagen (10). In brief, 0.2~1ug total RNA was poly-adenylated and used for cDNA synthesis primed by oligo-dT primer having a tail sequence recognized by an universal primer. cDNA was amplified using the miRNA specific and universal primers. 18S RNA served as internal controls (Ambion). PCR primer sequences are below. RT-PCR Ybx1 5’-GGATGGCAATGAAGAGGACAAA-3’ 5’-CGTCTGCGTCGGTAATTGAAGT-3’. RT-PCR Tsc-22

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5’-AAGCCATTTGATGTATGCGG-3’ 5’-GATTGTTCTCCTGCTCCAGCT-3’ Immunohistochemistry-Immunohistochemistry of renal cortical sections was performed as reported (10). 100 x dilution of Ybx1 (Abcam Inc.) or TSC-22 antibody (R&D Systems, Inc.) was used. Ybx1 and TSC-22 stained areas in glomeruli were quantified using Image-Pro Plus 5.1 software (Media Cybernetics, Inc.). Immunofluorescence- For Processing Body (P-body) detection, MMC were transfected with pcNEGFP-DCP1a (38) using a Nucleofector (LONZA). Cellular localization of P-bodies was detected by fluorescent microscopy. Immunofluorescence was performed as described (39). Briefly, cells grown on coverslips were fixed with 3% paraformaldehyde, permeabilized in 0.02% Triton-X-100, blocked with 1% BSA in PBS and immunostained using primary antibodies against Ybx1, and secondary antibodies conjugated with Texas Red. To detect the nuclei, cells were stained with 4,6-diamidino-phenylindole (DAPI)(Sigma). Cells were visualized through a 60x objective using appropriate filters on an Olympus BX51 fluorescence microscope (Olympus America). Separate images for each color were captured with a Pixera 600 CL camera using Viewfinder V3.0 software (Olympus), merged and processed using Adobe Photoshop 7.0. Western blot analysis- This was performed as described (9,10). Antibodies against YBX1 (Abcam Inc.), Phospho-YBX1, Akt, Phospho-Akt and actin (Cell Signaling), TFE3 (BD Bioscience), TSC-22 (R&D Systems, Inc.) were used. All primary antibodies were used at 1:1000 dilution. Cloning of the mouse Col1a2 proximal promoter- The proximal promoter of mouse Col1a2 gene was cloned by PCR using the primers below. The amplified fragment was cloned into pGL4 (Promega) and sequence of the insert confirmed. Humanized mouse Col1a2 proximal promoter was constructed by quick mutagenesis (Stratagene). For mutagenesis, PCR was performed using Pfu-ultra polymerase and primers below.

Primers for cloning the proximal promoter of mouse Col1a2 (lower case denotes sequences added for cloning) 5’-gcacagatctCCCACGTAGGTGTCCTAAAGTGC-3’ 5’-cgactaagcttACCGTTCCCTCCAACTTAGTCG-3’ Mutagenesis primers to introduce E-box into the proximal promoter of mouse Col1a2 gene 5’-CCCGCAGTCTCCTCCCAGCTGTGAGTCCCGGGCCC-3’ 5’-GGGCCCGGGACTCACAGCTGGGAGGAGACTGCGGG-3’ Potential E-boxes in the primers are underlined. Chromatin immunoprecipitation (ChIP) assays- ChIP assays were performed as reported (10). Briefly, MMC were serum-depleted and treated with TGF-β for 24 hr and then formaldehyde-fixed. The crosslinked chromatin was sheared and immunoprecipitated using antibodies against Tfe3, Tsc-22 or IgG (negative control). ChIP-enriched DNA was purified and used as template for real time qPCRs using primers spanning E-box regions as described (9). The proximal promoter region was amplified with the same primers used for cloning the Col1a2 promoter. Plasmids and Ybx1 3’-UTR reporters-Mouse Ybx1 3’UTR region was amplified by PCR using primers below, and high fidelity polymerase Pfu-ultra polymerase and digested with XhoI and NotI, and cloned into the NotI-XhoI site of psiCheck2 (Promega). Plasmid containing mouse Ybx1-3’UTR sequence was used as a reporter (psiCheck2mYbx1-3’UTR) while the plasmid containing the 3’UTR sequence harboring a mutation in the miR-216a site was a negative control (psiCheck2mYbx1-3’UTR-Mut). For mutagenesis of the miR-216a target site, two primers below were used. Tfe3 and its dominant negative mutant and shRNA expression vectors were described (10). Primers for PCR of Ybx1 3’UTR region 5’-GTACTCGAGTGCCGGCTTACCATCTCTAC-3’

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5’-ACTGCGGCCGCCTTTATTAACAGGTGCTTGCAGT-3’ miR-216a site mutagenesis primers into Ybx1 3’UTR 5’-CATATCTGGTCAAGTTCTCGAGTTTAAGAA-3’ 5’-TTCTTAAACTCGAGAACTTGACCAGATATG-3’

Luciferase assays- MMC were transfected with plasmids and/or small RNAs using Nucleofector as described (10). After 24 hours, Luc activities were measured as described (9). For miRNA and shRNA experiments, cells were treated for 48 hours. ShRNA against mouse Ybx1- U6 promoter-driven shRNA expression vector targeting Ybx1 was constructed as described (9). Briefly, U6 promoter region was amplified using 5’ U6 primer and Ybx1 target reverse primer below. The amplified fragment was cloned into pCR3.1EGFP while plasmid expressing scrambled shRNA (pCR3.1EGFP-Scr) was used as control. Ybx1 shRNA reverse primer 5’-GGACTCGAGAAAAAAGAAAACCTTCGTTGCGATGACCCTACACAAAGGTCATCGCAACGAAGGTTTTCGGTGTTTCGTCCTTTCC-3’ The target sequence is underlined. miRNAs and siRNA olgonucleotides- Oligonucleotides (oligos) representing the miR mimic, Negative Control #1, miR-216a inhibitor (2'-O-methyl modified RNA oligos designed antisense to the mature miR), Negative Control #1 for the inhibitor and TSC-22 siRNA pool were all obtained from Dharmacon as described (10).

Immunoprecipitation of RNA-protein complex and subsequent PCR- Ybx1-mRNA complexes were immunoprecipitated as described (40,41). After treatment with TGF-β, MMC were washed with cold PBS and lysed in buffer containing 100mM KCl, 5mM MgCl2, 10mM Hepes (pH 7.0), 0.5% Nonidet P-40 (NP40), 1mM DTT, 100 units/ml RNase inhibitor (Roche), 0.2mM PMSF and proteinase inhibitor cocktail Complete (Roche) and stored

at -80C. For immunoprecipitation, Protein-A Sepharose beads (Promega) were swollen in NT2 buffer containing 50mM Tris-pH7.4, 150mM NaCl, 1mM MgCl2, 0.05% NP40 and 5% BSA. 300 µl of protein-A bead slurry was incubated overnight at 4°C with 20 µl of Ybx1 antibody. The antibody-coated beads were washed with ice-cold NT2 buffer and resuspended in 900µl of NT2 buffer with100 units/ml RNase inhibitor, 1 mM DTT, 20 mM EDTA. The beads were mixed well and 100 µl of cell lysate was added. 100µl aliquot was removed for RNA sample without IP (Input). The remaining mixture (900ul) was rocked at room temperature for 2 hr and then washed 4 times with ice-cold NT2 buffer followed by 2 washes with NT2 buffer with 1M urea, re-suspended in 100 µl NT2 buffer with

0.1% SDS and 0.3mg/ml proteinase K and incubated for 30 min at 55°C. RNAs were extracted by phenol-chloroform and precipitated with ethanol. RT-PCR was performed as described (9). Protein domain structure prediction-For prediction of domain structures of TFE3 and TSC-22, on-line database SWISS-MODEL (http://swissmodel.expasy.org) (42) was used. Downloaded results were further analyzed using 3D Molecular Viewer (Vector NTI, Invitrogen) to depict interaction of two proteins through LZ structure.

Co-immunoprecipitaton (Co-IP)- Interaction between Tsc-22 and Tfe3 was examined by Co-IP. MMC treated with TGF-β were lysed in TNE buffer, 10mM Tris-HCl (pH7.4), 1% NP40, 1mM EDTA, pre-cleared with Protein-A beads for 1hr, immunoprecipitated with Tsc-22 antibody (goat IgG) or Tfe3 antibody (mouse IgG) overnight, washed 4 times with TNE buffer and eluted with SDS sample buffer. Immunoprecipitates were analyzed by Western blotting using reciprocal antibodies as mentioned above.

RESULTS and DISCUSSION

Decreased Ybx1 levels in mouse MC (MMC) treated with TGF-β and in diabetic kidney glomerul- TGF-β treatment led to a significant decrease in Ybx1 mRNA (from 6 to 24hr) and protein levels at 24 hr in MMC (Fig. 1A & B). Ybx1 mRNA levels were also

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significantly lower in kidney glomeruli from type 1 diabetic mice (streptozotocin [STZ] injected) (Figure 1C) as well as type 2 diabetic mice (db/db) (Fig. 1D) compared to their respective controls (control C57BL/6 and db/+). Parallel decrease in Ybx1 expression in glomeruli of these diabetic mice by immunostaining was observed compared to their non-diabetic controls (Fig. 1E-J). Ybx1 was suggested to be a potential target of miR-216a (43), and according to databases, miRBase (http://microrna.sanger.ac.uk), TargetScan (http://www.targetscan.org), microRNA.org (http://www.microrna.org/microrna/home.do) (Fig. 1K). We confirmed a significant increase in miR-216a levels in glomeruli of type1 and type 2 diabetic mice compared to corresponding control mice (Fig. 1L & M) as reported previously (10). TGF-β and Col1a2 gene expression levels were also increased in glomeruli from these diabetic mice (9). miR-216a levels were increased from 6 to 24 hour (2-3.5 fold) in MMC treated with TGF-β (Fig. 1N). Under both conditions (diabetic glomeruli and MMC treated with TGF-β), increased miR-216a and decreased Ybx1 levels were observed. These results suggest that increased miR-216a under diabetic conditions may inhibit the expression of its target, Ybx1.

Since TGF-β activates Akt (10,18,19) and Akt phosphorylates Ybx1 (40,44), phosphorylation of Ybx1 was examined (Fig.1B). Increased Ybx1 phosphorylation was observed at 6hr after TGF-β treatment but this disappeared by 24hr, while Akt activation (p-Akt) was sustained through 6-24 hr. Ybx1 is phosphorylated by TGF-β-activated Akt (at 6hr), however, by 24 hr, increased miR-216a subsequently reduces its target, Ybx1 protein. Therefore, Ybx1 is regulated by at least two different mechanisms by TGF-β. miR-216a inhibits Ybx1 in mouse kidney cells- In order to test if exogenous miR-216a addition inhibits endogenous levels of Ybx1, miR-216a mimic oligonucleotides (oligos) were transfected into MMC using nucleofection (10) and protein levels of Ybx1 were examined. Significant decrease of Ybx1 was observed in MMC transfected with miR-216a mimic in a dose-dependent manner (Fig. 2A&B).

Reciprocally, Col1a2 mRNA levels were increased by miR-216a mimic or by Ybx1 shRNA compared to their respective controls (NC and SCR) (Fig. 2C&D). We confirmed these results using a mouse kidney cell line, TCMK-1 cells. Ybx1 protein levels were decreased by over 80 % by the miR-216a mimic, while Ybx1 mRNA levels were not altered (Supplementary information Fig. S1A & B). In parallel, Col1a2 mRNA levels were increased by miR-216a mimic (Fig. S1C). mRNA and protein levels of Ybx1 were significantly reduced by Ybx1 shRNA (Fig. S1D & E), while Col1a2 mRNA levels were increased (Fig. S1F). These results strongly suggest that miR-216a targets endogenous Ybx1, and that Ybx1 is a key negative regulator of Col1a2 in mouse kidney MC. Ybx1 3’UTR is a direct target of miR-216a- To verify whether the Ybx1 3’UTR is a direct target of miR-216a, luciferase (Luc) reporter vectors containing the Ybx1 3’UTR were constructed (Fig. 2E). miR-216a mimic significantly inhibited Luc activity of Ybx1 3’UTR vector by almost 70% relative to a negative control Mimic (NC), but not in the two control constructs, namely control without 3’UTR (first pair of bars) or that with Ybx1 3’UTR that has base-substitutions in miR-216a site (third pair of bars) (Fig. 2F). Since TGF-β increased miR-216a expression (Fig. 1N), we tested the effects of TGF-β on the Ybx1 3’UTR reporter activity. TGF-β significantly inhibited Ybx1 3’UTR vector Luc activity in MMC (by almost 80% relative to untreated cells), but had no significant effect in control or miR-216a site mutant vector-transfected cells (Fig. 2G). These results suggest that miR-216a increased by TGF-β can inhibit Ybx1 3’UTR Luc activity. Then, we examined whether a miR-216a inhibitor reverses the effects in this system. miR-216 inhibitor demonstrated a dose-dependent increase in Luc activity from 5 to 20nM (Fig. 2H), further confirming Ybx1 as a true target of miR-216a. Mouse Col1a2 proximal promoter- Ybx1 was initially identified as a transcriptional factor. A potential Ybx1 binding sequence is present in the proximal promoter of human COL1A2 gene along with a Smad binding element (SBE, CAGA repeats). To evaluate a

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potential role in the mouse, we cloned the proximal promoter of the mouse Col1a2 gene. Consensus SBE (CAGA repeat) and a potential Y-box were found in this promoter region (Fig. 3A & B). However, this mouse proximal promoter did not respond to TGF-β (Fig. 3C, last set of bars), although the human COL1A2 proximal promoter was sufficient to respond to TGF-β (45,46). Since our previous data showed that E-boxes in far-upstream region of the mouse Col1a2 gene responds to TGF-β (9), we cloned these E-boxes into the mouse proximal promoter. This reporter responded to TGF-β (Fig. 3C, middle set of bars), demonstrating that E-boxes are essential for TGF-β response in mouse cells. To further investigate this discrepancy, we compared the sequences of human and mouse proximal promoters and found that the human promoter has a putative E-box (CAGCTG) while the mouse Col1a2 promoter has a two-base substitution (CAGCac) (Fig. 3A). This human E-box sequence was conserved in chimpanzee, monkey and pig but not in mouse or rat (Fig. 3A). Using quick mutagenesis method, we altered the sequence to introduce the E-box in the mouse proximal promoter. As expected, this humanized (+E-box) mouse proximal promoter of Col1a2 responded to TGF-β (Fig. 3C, first set of bars). These results demonstrate that the E-box is essential for TGF-β response and the mouse proximal promoter cannot respond to TGF-β because of the sequence substitution in the potential E-box. Collaboration of far-upstream enhancer E-boxes with the mouse Col1a2 proximal promoter is necessary for TGF-β response in mouse kidney cells.

E-boxes are essential for TGF-β response in the promoters of PAI-1, Smad7 and non-coding RNA RP23-298H6.1-001 (RP23) hosting miR-216a (3,10,47). These data are consistent with previous observations that the far-upstream enhancer region (including E-boxes) is necessary for strong expression of Col1a2 reporter gene in transgenic mice (48) and far-upstream E-boxes are major regulators of the mouse Col1a2 gene (4,5,9,10). This sequence variation in the Col1a2 gene between human and mouse or rat is intriguing and might explain the difference in their susceptibilities to diabetic kidney diseases.

Tfe3 is a positive regulator of E-boxes in the mouse Col1a2 promoter in response to TGF-β- Our data show that E-boxes play key roles in Col1a2 promoter activity in response to TGF-β in MMC. E-box repressors, such as Zeb1 and Zeb2, act as negative regulators of the Col1a2 gene (4,9,10). However, positive regulators of E-boxes in the Col1a2 gene promoter have not yet been described. In TGF-β signaling, Tfe3 is a major transcription factor associated with the expression of the PAI-1, Smad7 and RP23 (host gene for miR-216a) genes (3,10,47). Tfe3 is also a positive regulator of E-boxes in the IRS2 promoter (11). Therefore, we next tested whether Tfe3 is a candidate positive regulator of Col1a2 gene expression. Tfe3 protein expression was increased by TGF-β in a dose- (5-10 ng/ml) and time-dependent (6 to 24 hr) manner in MMC (Fig. 4A). Chromatin immunoprecipitation (ChIP) assays showed that the occupancy of Tfe3 at E-boxes in the far-upstream enhancer region of the Col1a2 gene was significantly increased at 6 hr post TGF-β treatment with 10-fold enrichment at 24 hr (Fig. 4B & C). The occupancy of the E-box repressor Zeb1 at this same E-box in the Col1a2 enhancer was decreased by over 10-fold by TGF-β (Fig. 4D), as noted earlier (9). These results demonstrate that the E-box repressor Zeb1 is replaced by the activator Tfe3 in response to TGF-β.

To further verify the positive role of Tfe3 on the Col1a2 promoter, a Tfe3 expression vector was co-transfected into MMC with two types of Col1a2 promoter reporter constructs. Tfe3 overexpression had no effect on the Col1a2 promoter without E-box (Col1a2P-luc) (Fig. 4E) (left set of bars), even after TGF-β treatment. However, Tfe3 increased the basal reporter activity of a Col1a2 promoter containing E-boxes (E-box-Col1a2P-luc) and its induction by TGF-β significantly (Fig. 4E) (right set of bars). On the other hand, a dominant negative Tfe3 mutant or Tfe3 siRNA clearly inhibited basal and TGF-β induced Luc activity of the same construct (E-box-Col1a2P-luc) (Fig. 4F). These results further demonstrate that Tfe3 is a major positive regulator of the Col1a2 promoter at E-boxes and mediates TGF-β responses in MC.

In current studies, we could not demonstrate any role for the potential Y-box in

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mouse proximal promoter in the TGF-β response. No change in promoter activity or TGF-β response was detected when the Y-box was deleted from the proximal promoter constructs, even when the far-upstream E-boxes were present (Fig. 3C). Therefore, this potential Y-box might play a role in basal Col1a2 expression. We therefore next pursued other potential mechanisms by which Ybx1 might regulate Col1a2 gene under our experimental conditions. Colocalization of Ybx1 with P-bodies in MMC- Ybx1 is also known as an RNA binding protein that regulates mRNA stability and translation (40,49,50). Ybx1 was reported to be a component of cytoplasmic Processing-bodies (P-bodies) and stress granules (51,52). Ybx1 was also found in the Ago-binding protein complexes (53). Therefore, we examined the intracellular localization of Ybx1 in MMC. MMC were transfected with pcNEGFP-DCP1a, a well-known P-body marker (38) and Ybx1 localization was detected by immunofluorescence. DCP1aGFP signals (green spots) were observed in the cytoplasmic region of MMC (Fig. 5A) indicating P-bodies. Ybx1 (Texas-red staining) was located in both nuclear and cytoplasmic regions, and many of the Ybx1 stained spots clearly overlapped with green DCP1a spots (Fig. 5B&C, arrows), suggesting that Ybx1 and P-bodies were clearly, but partially, colocalized. Interestingly, 24 hr after TGF-β treatment, Ybx1 spots disappeared and cytoplasmic Ybx1 staining was greatly attenuated (Fig. 5K and 5L, TGF-β 24 hr) while no change in control untreated cells (Fig. 5E or 5B and 5F or 5C respectively) or to 6hr TGF-β treatment (Fig. 5H & 5I). miR-216a mimic also induced a decrease in cytoplasmic Ybx1 signals (Fig. 5Q & 5R), similar to TGF-β actions, compared to negative control mimic treatment (Fig. 5N &5O). Notably, Ybx1 signals in P-body (GFP)-positive cells were clearly weaker than in those without P-body signals (Fig. 5P-R) while no difference was observed between GFP-positive and negative cells transfected with NC (Fig. 5 M-O), indicating that cells transfected with miR-216a (GFP-positive) have less levels of Ybx1 protein. These results are consistent with the data in Fig. 1 and 2 showing that TGF-β

or miR-216a inhibits Ybx1 expression in MMC. No clear change in P-bodies themselves was detected even after treatment with TGF-β or miR-216a mimic relative to corresponding controls (Fig. 5A, D, G, J, M, P), suggesting that TGF-β and miR-216a do not directly affect P-bodies and that Ybx1 may function as an RNA binding protein in cytoplasmic P-bodies in response to TGF-β or miR-216a. Ybx1 plays a role as an RNA binding protein in response to TGF-β- A series of transcripts have been reported to be Ybx1-binding RNAs (40). We first tested whether Col1a2 mRNA binds to Ybx1 in MMC using RNA immunoprecipation (RNA-IP). However, Col1a2 mRNA was not precipitated with Ybx1 (Fig. 6A), although we did detect Tsc-22 (TGF-β inducible clone 22 or TGFβi4) transcripts, a reported positive control for this RNA-IP experiment (40). However, interestingly, Ybx1-Tsc-22 mRNA binding was greatly decreased after TGF-β treatment (Fig. 6A). qPCR further confirmed that the relative amount of Tsc-22 RNA bound to Ybx1 was significantly decreased by TGF-β (Fig. 6B). Conversely, Tsc-22 protein levels were increased by TGF-β in MMC (Fig. 6C), suggesting an enhanced translation of Tsc-22 after release of the Tsc-22 mRNA from Ybx1. Ybx1 shRNA or miR-216a mimic reduced the interaction of Tsc-22 mRNA with Ybx1 while the miR-216a inhibitor increased the interaction (Fig. 6D-F). These results suggest that TGF-β may enhance Tsc-22 translation through upregulation of miR-216a which targets and reduces Ybx1 (Fig. 6G). Tsc-22 levels were also increased in the glomeruli of diabetic db/db mice compared to db/+ mice (Fig. 6H-J). Furthermore, Tsc-22 siRNA inhibited TGF-β-induced Col1a2 in MMC (Fig. 6K&L). These data suggest that miR-216a can reduce its target Ybx1 and the amount of Tsc-22 mRNA bound to Ybx1, and thereby increase Tsc-22 translation to augment TGF-β induced collagen expression (Fig. 6G & Supplementary Fig. S2). Tsc-22 interaction with Tfe3 on far-upstream E-box region enhances Col1a2 expression in response to TGF-β- In order to examine how Tsc-22 upregulates Col1a2, we next performed ChIP assays on the far-upstream E-box region and proximal promoter of the

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Col1a2 gene (Fig. 7A-D). Occupancy of Tsc-22 at the E-boxes was increased at 24hr after TGF-β treatment, with no change on the proximal promoter region. No change in Smad4 occupancy at either of these regions was observed, although a report showed that Tsc-22 enhances TGF-β signaling through Smad4 (54). This could be due to the fact that Smad proteins are expressed at relatively low levels in adult human and mouse kidney mesangium (55). The contribution of Smad proteins to TGF-β signaling in the mesangium may be relatively less than non-Smad and E-box dependent mechanisms.

Since we found Tfe3 enhanced Col1a2 expression through E-boxes (Fig. 4), we examined the domain structures of Tfe3 and Tsc-22 for potential interaction using the database, Swiss-Model (http://swissmodel.expasy.org) (42). Interestingly mouse Tsc-22 has a leucine zipper (LZ) structure in its carboxyl terminus (992-1047) while mouse Tfe3 has a helix-loop-helix leucine zipper (HLH-LZ) structure in the middle (205-289) (Fig. 7E, Supplementary Fig. S3). These two domains could potentially interact with each other through the LZ (Fig. 7F and Fig. S3). These structures and potential interactions are conserved in human TFE3 and TSC-22 (Supplementary Fig. S4). To test the interaction between Tfe3 and Tsc-22, we performed Co-IP experiments (Fig. 7G-I). No signal was detected in serum depleted (SD) untreated MMC (Fig. 7H-I) due to very low basal expression of these two proteins (Fig. 4A & 6C). However, clear interaction between Tfe3 and Tsc-22 was detected in lysates from MMC treated with TGF-β. Tfe3 was detected in lysates immunoprecipitated with Tsc-22 and vice versa relative to IgG control (Fig. 7G-I). In addition, Tsc-22 siRNA inhibited the TGF-β-induced increase in Col1a2 E-box Luc reporter activity (Fig. 7J). The results suggest a novel function of Tsc-22 in response to TGF-β in enhancing Col1a2 expression by collaborating with Tfe3 at the far-upstream E-box region (Fig. 7K).

It should be noted that Upstream Stimulatory Factors (USF1 and USF2) have been reported to be involved in TGF-β upregulation by high glucose (HG) and diabetic conditions and associated with the pathogenesis

of DN (13-15,56). USFs are also E-box-regulators and have LZ motif which is critical for homo- or hetero-dimerization (16). USFs also mediate TGF-β-induced plasminogen activator inhibitor-1 (PAI-1), collaborating with EGFR signaling (17). Therefore, it is possible USFs may also be involved in TGF-β-induced Col1a2 in MMC by interacting with Tsc-22 through their LZ motifs. This is intriguing and remains to be elucidated in the future. Collagen induction by diabetic conditions and TGF-β mediated by miR-216a, Ybx1, Tsc-22 and Tfe3- Our results demonstrate that miR-216a upregulated by diabetic conditions or TGF-β targets Ybx1, an RNA-binding protein. Ybx1 reduction releases Tsc-22 mRNA (probably from P-bodies) to enhance Tsc-22 protein levels in MMC treated with TGF-β. Tsc-22 in turn interacts with Tfe3 and enhances Col1a2 expression through far-upstream E-box region (Supplementary Fig. S2). TSC-22 was cloned as a gene induced by TGF-β (57) and regulated post-transcriptionally by TGF-β (58). Interestingly, a specific allele in the promoter region of TSC-22 gene is associated with an increased risk of human DN (59), suggesting that TSC-22 plays a role in the development of human DN.

On the other hand, Ybx1 is also phosphorylated by Akt and this promotes the translation of mRNAs bound to Ybx1 (40,44,60). In the current study, we also observed increased Akt activation and Ybx1 phosphorylation in TGF-β-treated MMC (Fig. 1B). Therefore, Ybx1 may be regulated by two different miRNA-regulated mechanisms in response to TGF-β. One is via phosphorylation and inactivation of Ybx1 by TGF-β-induced Akt activation (via inhibition of Pten targeted by miR-216a)(10,19). The other is via downregulation of Ybx1 targeted by miR-216a. Both steps are mediated by miR-216a induced by TGF-β.

The promoter of the non-coding RNA RP23 (the host gene of miR-216a and miR-217) also contains E-boxes (10), suggesting that, apart from the Co1a2 promoter, increased miR-216a may also activate its own promoter through Tsc-22 and Tfe3. Therefore, the miR-216a/Ybx1 pathway can amplify the signaling initiated by TGF-β (Supplementary Fig. S2). These positive

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feed-back signaling loops can further accelerate and augment signaling through miR-216a and miR-217 and their targets Ybx1 and Pten. Therefore, once the signal is initiated by TGF-β, these downstream signaling pathways (including miRNAs, Ybx1 and Pten and Akt kinase) are amplified. This could be a major mechanism for chronic renal diseases such as DN.

In summary, the current results demonstrate that post-transcriptional regulation

by Ybx1, a target of miR-216a which is induced by TGF-β in MMC, is a key mechanism for enhanced mRNA translation under diabetic conditions. This novel regulation linked with enhanced accumulation of ECM proteins such as collagen may be involved in pathological sclerosis associated with not only diabetic kidney disease but also other fibrotic disorders and wound healing.

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FOOTNOTES *This work was supported by Grants from the National Institutes of Health (NIH NIDDK R21 DK076669, R01DK58191 and R01 DK081705). The authors are grateful to all members of the Natarajan laboratory for helpful discussions, Jane Zhang and Indu Nair for assistance, Hitoshi Shimano and Yoshimi Nakagawa for Tfe3 plasmids and Jens Lykke-Andersen for pcNEGFP-DCP1a. The authors declare that they have no conflict of interest.

FIGURE LEGENDS Figure 1. Expression of Ybx1 and miR-216a. (A) Ybx1 mRNA levels were significantly decreased in MMC treated with TGF-β (10ng/ml). Bars represent the mean ± SEM (n=3). (B) Ybx1 protein levels were decreased by 24 hr in MMC treated with TGF-β. Phosphorylation of Ybx1 (P-Ybx1) was detected at 6hr but disappeared at 24 hr. Increased Akt phosphorylation was observed at 6 -24hr. (C & D) mRNA expression levels of Ybx1 in mouse glomeruli. Significant decrease of Ybx1 mRNA levels in glomeruli from both type 1 and 2 diabetic mice compared to control mice. mean ± SEM (n=4). (E-J) Immunohistochemical staining of Ybx1 in glomeruli from normal and diabetic mice. Significant decrease in Ybx1 staining in glomeruli of type 1 (STZ) diabetic (F) compared to control (CTR) mice (E) (Scale bar, 20μm) and in db/db type 2 diabetic (H) compared to control db/+ mice (G). (I & J) Significant decrease of Ybx1 staining in diabetic mouse kidney glomeruli (STZ or db/db), compared to control (CTR or db/+). mean ±SEM (n=3). (K) Alignments of human and mouse miR-216a and Ybx1 3’UTR sequences. (L-N) miR-216a expression levels are increased in glomeruli from type 1 (STZ) diabetic mice (L) and from type 2 (db/db) diabetic mice (M) and in MMC treated with TGF-β (N) compared to respective controls. mean ± SEM (n=3). ***, ** and * indicate P<0.001, P<0.01 and P<0.05, respectively. Figure 2. Ybx1 is a target of miR-216a. (A & B) Ybx1 protein expression in MMC after transfection with miR-216a Mimic. Ybx1 protein levels were significantly decreased by 10-20 nM miR-216a mimic. mean ± SEM (n=4). (C) Significant increase of Col1a2 mRNA levels by miR-216a mimic relative to NC. mean ± SEM (n=3). (D) Significant increase of Col1a2 mRNA levels by Ybx1 shRNA relative to Scrambled control (SCR). mean ± SEM (n=3). (E) Luciferase (Luc) reporters for mouse Ybx1 3’UTR. Plasmid containing Luc and Ybx1 3’UTR (miR-216a

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target site) was used as reporter of miR-216a (Ybx1 3’UTR). Reporters without 3’UTR (Control, psiCheck2, Promega) or a miR-216a site mutant in Ybx1 3’UTR (Ybx1-3UTR-MT) were used as controls. (F) Significant decrease in Luc activity with Ybx1 3’UTR by niR-216a but not with other control reporters. (G) Significant decrease in Luc activity with TGF-β in Ybx1 3’UTR reporter, with no change in the negative controls. (H) Significant increase of Luc activity by miR-216a inhibitor in a dose-dependent manner. mean ± SEM (n=4). ***, ** and * indicate P<0.001, P<0.01 and P<0.05, respectively. Figure 3. Col1a2 proximal promoter activity. (A) Comparison of the proximal promoter sequence of the Col1a2 gene in several species (human, chimpanzee, monkey, pig, rat and mouse). Conserved consensus sequences of potential Smad site (CAGA repeats), Y-box, and TATA box (TA) were found in all the listed species. Potential E-box (CAGCTG) was found in the promoter of human, chimpanzee, monkey and pig. However, two base-substitutions that disrupted the E-box consensus were in rat and mouse (CAGCac). (B) Structure of the promoter region of mouse Col1a2 gene. A cluster of three E-boxes is located in the 16kb far-upstream region of mouse Col1a2 gene. (C) Response of the mouse proximal promoter to TGF-β. The cloned proximal promoter of mouse Col1a2 gene did not respond to TGF-β (bottom construct). Addition of far-upstream E-boxes conferred TGF-β response to this mouse proximal promoter (third construct). Deletion of the Y-box and Smad sites led to loss of basal promoter activity (fourth construct) even if the upstream E-boxes were added (second construct). Base-substitutions which created a putative E-box (the same sequence as the human promoter, humanized) restored the TGF-β response (top construct). mean ± SEM (n=4). *** and * indicate P<0.001 and P<0.05, respectively. Figure 4. E-box binding factors are major regulators of the mouse Col1a2 gene promoter region. (A) Dose-response and time course effect of TGF-β on Tfe3 protein levels in MMC by Western blot analysis. Tfe3 expression levels were increased by 5ng/ml and 6hours of TGF-β. (B) ChIP analysis of Tfe3 on the far-upstream E-box region of mouse Col1a2 gene. Regular PCR results of ChIP enriched DNA (top panel). Schematic structure of upstream of Col1a2 gene (bottom panel). Three E-boxes are located 16kb upstream as described previously and arrows are primers used for ChIP PCR (9). (C) Significant increase of Tfe3 occupancy at the Col1a2 enhancer (E-box region) at 6-24 hr post TGF-β treatment (n=3). (D) Combined results of occupancies of Zeb1 and Tfe3 on Col1a2 gene enhancer. Zeb1 occupancy was significantly decreased by TGF-β while Tfe3 was increased. mean ± SEM (n=3) (ChIP-qPCRs) (E) Roles of E-box and Tfe3 on activity of the mouse Col1a2 proximal promoter. The proximal promoter without upstream E-boxes (Col1a2P-luc) did not respond to TGF-β or to Tfe3 overexpression. However, the promoter containing three E-boxes (E-box-Col1a2P-luc) responded to TGF-β and Tfe3 suggesting that E-boxes located in far-upstream of Col1a2 gene are essential for TGF-β response. (F) Effects of dominant negative (dn) Tfe3 and Tfe3 siRNA on the mouse Col1a2 promoter (with E-boxes). dn Tfe3 or Tfe3 siRNA inhibited basal and TGF-β-induced reporter activity of E-box-Col1a2P-luc. mean ± SEM (n=4). ** and * indicate P<0.01 and P<0.05, respectively. Figure 5. Colocalization of Ybx1 with P-bodies and reduction of Ybx1 by TGF-β or miR-216a in MMC. P-bodies (green spots) were visualized by transfecting MMC with pDCP1-EGFP under fluorescence microscopy (left panels). Ybx1 spots and staining (red) were visualized by immuno-fluorescence using Ybx1 antibody and Texas-red-conjugated secondary antibody (middle panels). Right panels show merged images of GFP and Texas-red signals. Nuclei were stained with DAPI (blue). Under control conditions (serum depleted) (A-F) (A-C are enlarged images of D-F), Ybx1 spots (B) as well as P-bodies (A) were observed in cytoplasmic regions. Scale bar, 20μm for (A) and 50μm for (D). Cytoplasmic Ybx1 spots and staining were detectable 6hr after TGF-β treatment (H), but markedly reduced at 24 hr (K). miR-216a mimic treatment also markedly decreased Ybx1 signals (Q) compared to negative control (NC) (N). In miR-216a transfection experiments, Ybx1 signals were weaker in GFP positive cells compared to GFP-negative cells (P versus Q), further confirming that miR-216a inhibits Ybx1 expression

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(Q). P-body signals were stable even after TGF-β or miR-216a treatment (A, D, G, J, M, P). Merged images of green signals (DCP1-EGFP) and red signals (Ybx1, Texas-red) shown in (C, F, I, L, O, R). Figure 6. Ybx1 functions as an RNA-binding protein. (A) Tsc-22 mRNA precipitated with Ybx1 but not Col1a2 mRNA. TGF-β decreased Ybx1-Tsc-22 mRNA binding. M, molecular weight markers. IP, immunoprecipitation. Input, RNA extracted from RNA-protein complex without IP. (B) Real time qPCR of RNA-IP samples. TGF-β decreased Ybx1-Tsc-22 mRNA interaction significantly. Ybx1 RNP, Ybx1-containing ribonucleoprotein. mean ± SEM (n=3). (C) Tsc-22 protein levels were increased at 24 hour after TGF-β treatment. (D & E) Ybx1 shRNA (D) and miR216a mimic (E) significantly decreased Tsc-22 mRNA levels in Ybx1 RNP. (F) miR-216a inhibitor significantly increased Tsc-22 mRNA levels in Ybx1 RNP. (G) Schematic model of the upregulation of Tsc-22 translation by TGF-β or diabetic conditions. Ybx1 decrease by miR-216a releases Tsc-22 mRNA from P-body leading to enhancement of Tsc-22 translation. (H-J) Immunostaining of Tsc-22 in mouse renal glomeruli. Significant increase of Tsc-22 in glomeruli from db/db type 2 mice compared to control db/+ mice (Scale bar, 20μm). (K & L) Inhibition of Tsc-22 by Tsc-22 siRNA blocked TGF-β induced upregulation of Col1a2 in MMC. mean ± SEM (n=3). ***, ** and * indicate P<0.001, P<0.01 and P<0.05, respectively. Figure 7. Tsc-22 interacts with Tfe3 and enhances Col1a2 through far-upstream E-box region in response to TGF-β. (A) ChIP analysis of Tfe3 and Tsc-22 on Col1a2 far-upstream E-box region. Significant increase of occupancy of Tsc-22 at 24 hr of TGF-β treatment but no change of Smad4 occupancy. mean ± SEM (n=3). (B) No significant change of Tsc-22 or Tfe3 occupancy on the Col1a2 proximal promoter. (C) Combined results of occupancy of Zeb1, Tfe3, Tsc-22 and Smad4 on Col1a2 E-box region. Zeb1 occupancy was significantly decreased by TGF-β whereas Tfe3 and Tsc-22 occupancies were increased. No change of Smad4 occupancy observed. (D) Upstream promoter region of the Col1a2 gene. Arrows indicate positions of primers used for ChIP analysis. (E) Domain structures of mouse Tsc-22 and Tfe3. Leucine zipper (LZ) was found in the carboxyl terminus of Tsc-22 (upper). Helix-loop-helix leucine zipper (HLH-LZ) was in the central region of Tfe3 (lower). Domain structures were predicted using Swiss-Model (http://swissmodel.expasy.org) (42). (F) Potential interaction between two domains (Tsc-22 LZ and Tfe3 HPH-LZ) depicted by 3D Molecular Viewer. (G) Tsc-22 protein was detected in lysates immunoprecipitated with Tfe3 or Tsc-22 (IP). Input, 2.5% of protein lysate of MMC treated with TGF-β before IP. No band was detected in the IP sample with control IgG. WB, Western blot. (H) Tfe3 was detected in Tsc-22 IP sample from MMC treated with TGF-β. (I) Tsc-22 was detected in Tfe3 IP sample from MMC treated with TGF-β. (J) Tsc-22 siRNA pool inhibited TGF-β-induced increase in Col1a2 promoter reporter activity (+E-boxes). NC, negative control siRNA pool. mean ± SEM (n=4). ** and * indicate P<0.01 and P<0.05, respectively. (K) Schematic model of activation of Col1a2 promoter in response to TGF-β. In the absence of TGF-β, Zeb1 occupies the E-boxes at the far-upstream region of the Col1a2 gene and inhibit transcription. TGF-β treatment or diabetic conditions inhibit Zeb1, but increase Tfe3 and Tsc-22. Tfe3/Tsc-22 complex in turn bind to the E-boxes of the Col1a2 gene and enhance transcription.

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Figure 1

miR-216a & Ybx1

Target site alignment between Human and Mouse

3' GUGUCAAC--GGUCGACUCUAAU 5' Human hsa-miR-216a||:| || | ||:|||||||

278:5' CAUAUCUGGUCAAGUUGAGAUUU 3' Human Ybx1 3’UTR||||||||||||||||||||||| Conservation: 100.0%

274:5' CAUAUCUGGUCAAGUUGAGAUUU 3' Mouse Ybx1 3’UTR||:| || | ||:|||||||

3' GUGUCAAC--GGUCGACUCUAAU 5' Mouse mmu-miR-216a

Target site alignment between Human and Rat

3' GUGUCAAC--GGUCGACUCUAAU 5' Human hsa-miR-216a||:| || | ||:|||||||

278:5' CAUAUCUGGUCAAGUUGAGAUUU 3' Human Ybx1 3’UTR||||||||||||||||||||||| Conservation: 100.0%

169:5' CAUAUCUGGUCAAGUUGAGAUUU 3' Rat Ybx1 3’UTR||:| || | ||:|||||||

3' GUGUCAAC--GGUCGACUCUAAU 5' rno-miR-216a

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CTR 6hr 24hr

Expr

essi

onle

vels

***

TGF-β

A

miR-216a

00.51

1.52

2.53

db/+ db/db

*

glomeruli

MmiR-216a

CTR STZ0

0.20.40.60.81

1.21.41.61.8 *

glomeruli

L

Expr

essi

on le

vels

Ybx1

00.20.40.60.811.2

CTR STZ

Expr

essi

onle

vels

***

glomeruli

C Ybx1

00.20.40.60.81

1.2

db/+ db/db

**

glomeruli

D

Ybx1

012345678

CTR STZ

Ybx1

pos

itive

are

a (%

) **I

**Ybx1

012345678

db/+ db/db

J

-Ybx1

-actin

CTR 6hr 24hrTGF-βB

-P-Ybx1

-P-Akt-Akt

Control STZ db/+ db/db

Ybx1E F G H

14

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Figure 2

00.20.40.60.8

11.21.4

Control Ybx13UTR

Ybx13UTR-MT

Rel

ativ

e lu

cac

tivity

NoneTGF-β

*

GNC MimicmiR-216a Mimic

F

00.20.40.60.8

11.21.4

Control Ybx13UTR

Ybx13UTR-MT

Rel

ativ

e lu

cac

tivity *

Col1a2

00.20.40.60.81

1.21.41.61.82

NC miR-216a

Rel

ativ

e le

vels

*

CA

Actin-

Ybx1-

miR-216a Mimic

MMC

Western blot

0nM 10nM 20nM

*

**

miR-216a inhibitor

0123456

0nM 5nM 10nM 20nMconcentration

Rel

ativ

e lu

cac

tivity

H

*B

Ybx1/actin

00.2

0.40.60.8

11.2

0 10 20miR-216a Mimic (nM)

Nor

mal

ized

leve

lsle

vels

*** Col1a2

01234567

SCR Ybx1shRNA

Rel

ativ

e le

vels

*

D

15

SV40-P luciferase

E

Control

Ybx1 3’ UTRSV40-P luciferase

miR-216a

Ybx1-3UTR

SV40-P luciferase

x

Ybx1-3UTR-MTYbx1 3’ UTR

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CAGCac

Relative luc aitivityMouse Col1a2 proximal promoter

TGF-βNone

E E E LucTA

E E E Smad LucTA *

***Smad Y HuE LucTACAGCTG

Smad Y LucTACAGCac

C

LucTA

Col1a2 gene

A Comparison of Col1a2 proximal promoters

TATASmad Y-box E-box

TATA-boxTranscription start

-37

CCAATPotential Y-box

-191-206

Smad siteCAGA repeat -248-269 CAXXTG

Potential E-box

-116-125

Figure 3

E-box E-box E-box

Far upstream enhancer region

Col1a2 gene

~16kb

Proximal promoter

TATASmad Y-box

B Mouse Col1a2 promoter

0 1 2 3 4 5 6

Y

16

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A

-Tfe3

-actin

0 6 24 hrTGF-β (10ng/ml)

0 2 5 10 ng/ml

-actin-Tfe3

TGF-β (24 hour)

-ChIP DNA (αTfe3)-Input

0 6 24 hr TGF-β treatment

E-box E-box E-box

Col1a2 enhancer region

Col1a2 gene

~16kb

B

Tfe3ChIP Real time PCR

02468

10121416

0 6 24hrTGF-β treatment

Rel

ativ

e oc

cupa

ncy

**

*

C

Figure 4

Smad Y LucTA Col1a2P-luc

E-box-Col1a2P-lucE E E Smad Y LucTA

ChIP on Col1a2 E-box

0.01

0.1

1

10

100

0 6 24hrTGF-β treatment

Rel

ativ

e oc

cupa

ncy

Tfe3

Zeb1

D

*

*

**

**

NoneTGF-β

Col1a2P-luc E-box-Col1a2P-luc

*

* **

E

0

0.5

1

1.5

2

2.5

Tfe3- Tfe3+ Tfe3- Tfe3+

Rel

ativ

e lu

cac

tivity

E-box-Col1a2P-luc

EGFPSCR

Tfe3dn Tfe3 siRNA

NoneTGF-β

**

*

**

F

0

0.5

1

1.5

2

Rel

ativ

e lu

cac

tivity

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DCP1-EGFP Ybx1 (Texas-red)

Figure 5

AControl

18

Merge

CB

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G H I

TGF-β

24h

J K L

NC

M N O

miR

-216

a

P Q R

Con

trol

D E F

TGF-β

6h

DCP1-EGFP

19

Figure 5 (continued)

Ybx1 (Texas-red) Merge

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-Tsc-22

-Actin

0 6 24 hr TGF-β

Western blot

CFigure 6

M TGF-β- -+ +

αYbx1

Col1a2 Tsc-22

-Input

-IP

A

***Tsc-22 in Ybx1 RNP

00.20.40.60.81

1.2

TGF-β(-)

TGF-β(+)

Rel

ativ

e le

vels

B

*Tsc-22 in Ybx1 RNP

00.20.40.60.81

1.2

SCR Ybx1shRNA

Rel

ativ

e le

vels

D Tsc-22 in Ybx1 RNP

**

00.20.40.60.81

1.2

Mimic NC

miR-216a

Rel

ativ

e le

vels

E**

Tsc-22 in Ybx1 RNP

0

0.5

1

1.5

2

2.5

InhibitorNC

inhibitor 216a

Rel

ativ

e am

ount

F

Tsc-22

H Idb/+ db/db

J Tsc-22

0

5

10

15

20

25

db/+ db/dbTSC

-22

posi

tive

area

(%)

**

Tsc-22

00.20.40.60.81

1.21.4

NC Tsc-22 siRNA

Rel

ativ

e ex

pres

sion

leve

ls ***

K

Rel

ativ

e ex

pres

sion

leve

ls

**Col1a2

00.51

1.52

2.53

3.5

NC Tsc-22 siRNA

NoneTGF-β

**

L

Ybx1

Tsc-22 mRNA

P-body

Tsc-22 mRNA

P-body

Tsc-22 mRNA

Tsc-22

translation

miR-216aDiabetic conditionsTGF-β

ribosome

G

20

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E-box E-box E-box

Far upstream enhancer region

Col1a2 gene

~16kb

Proximal promoter

TATASmad Y-box

Col1a2 E-box

20

468

1012

0h 6h 24h 0h 6h 24h

Smad4 Tsc-22

*

Rel

ativ

e oc

cupa

ncy

Col1a2 proximal promoter

00.20.40.60.81

1.21.4

0h 6h 24h 0h 6h 24h

Smad4 Tsc-22

Col1a2 Ebox+PP Luc

00.20.40.60.81

1.21.41.6

None TGFβ

NCTsc-22 siRNA

* **

Rel

ativ

e lu

cac

tivity

Leucine

Tsc-22LZ (992-1047)

Tfe3 HLH LZ (205-289)

Mouse Tsc-22

992 1047

LZ

Mouse Tfe3

205-289

HLH LZ

A

E

F G

J

E-box Col1a2

Tfe3

E-box Col1a2

Zeb1

Tsc-22

Diabetic conditionsTGF-β

K

Figure 7

-Tsc-22 WB

IgG

Tfe3

Inpu

t (2.

5%)

Tsc-

22

IP

MMC (TGF-β)Ig

G

Tsc-

22

Tsc-

22Ig

GIP

-Tfe3 WB

SD TGF-β

MMC

IgG

Tfe3

Tfe3

IgGIP

-Tsc-22 WB

SD TGF-βMMC

B

D

H

I

Col1a2 E-box

0.01

0.1

1

10

100

0h 6h 24h

Rel

ativ

e oc

cupa

ncy

Tfe3

Zeb1

Smad4

Tsc-22

TGF-β treatment

C

*

*

***

**

21

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Lanting, Ivan Todorov, John J. Rossi and Rama NatarajanMitsuo Kato, Lin Wang, Sumanth Putta, Mei Wang, Hang Yuan, Guangdong Sun, Linda

-induced collagen expression in kidney cellsβTGF-Posttranscriptional upregulation of Tsc-22 by Ybx1, a target of miR-216a, mediates

published online August 16, 2010J. Biol. Chem. 

  10.1074/jbc.M110.165027Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2010/08/31/M110.165027.DC1

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