RNA-Binding Protein PTB and MicroRNA-221 Coregulate AdipoR1 Translation and Adiponectin Signaling

  1. Hannah Kanety1,3
  1. 1Institute of Endocrinology, Chaim Sheba Medical Center, Tel-Hashomer, Israel
  2. 2Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel
  3. 3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
  4. 4Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
  1. Corresponding author: Hannah Kanety, hkanety{at}sheba.health.gov.il, or Yaniv Lustig, yaniv.lustig{at}sheba.health.gov.il.

Abstract

Adiponectin receptor 1 (AdipoR1) mediates adiponectin’s pleiotropic effects in muscle and liver and plays an important role in the regulation of insulin resistance and diabetes. Here, we demonstrate a pivotal role for microRNA-221 (miR-221) and the RNA-binding protein polypyrimidine tract–binding protein (PTB) in posttranscriptional regulation of AdipoR1 during muscle differentiation and in obesity. RNA-immunoprecipitation and luciferase reporter assays illustrated that both PTB and miR-221 bind AdipoR1-3′UTR and cooperatively inhibit AdipoR1 translation. Depletion of PTB or miR-221 increased, while overexpression of these factors decreased, AdipoR1 protein synthesis in both muscle and liver cells. During myogenesis, downregulation of PTB and miR-221 robustly induced AdipoR1 translation, providing a mechanism for enhanced AdipoR1 protein expression and activation in differentiated muscle cells. In addition, since both PTB and miR-221 are upregulated in liver and muscle of genetic and dietary mouse models of obesity, this novel translational mechanism may be at least partly responsible for the reduction in AdipoR1 protein levels in obesity. These findings highlight the importance of translational control in regulating AdipoR1 protein expression and adiponectin signaling. Given that adiponectin is reduced in obesity, induction of AdipoR1 could potentially enhance adiponectin beneficial effects and ameliorate insulin resistance and diabetes.

Introduction

Adiponectin, an adipocyte-derived abundant plasma protein (14) with insulin-sensitizing and anti-inflammatory properties, gained recognition as a potential mediator between obesity, insulin resistance, and diabetes (5): Adiponectin levels are reduced in obesity (1), and mice lacking adiponectin develop insulin resistance, glucose intolerance, hyperglycemia, and hypertension—all characteristics of the metabolic syndrome (5,6). Adiponectin's biological effects depend not only on the relative circulating concentrations of the hormone but also on the expression level and function of its receptors (5,79). To date, two receptors for adiponectin have been identified (AdipoR1 and AdipoR2), which mediate adiponectin pleoitropic effects (10). Downregulation of the receptors in obesity is involved in the development of insulin resistance and diabetes (11).

Skeletal muscle and liver are important targets of adiponectin and its receptors in the regulation of energy metabolism (12). Adiponectin, through AdipoR1, activates AMP-activated protein kinase (AMPK) in vivo and in vitro, which increases fatty acid oxidation and glucose utilization and leads to an improvement in insulin sensitivity (10,11,1315). Recently, overexpression of AdipoR1 in rat skeletal muscle was shown to enhance insulin sensitivity in vivo through phosphatidylinositol 3-kinase– and AMPK-signaling pathways (16). In addition, specific deletion of AdipoR1 in mouse skeletal muscle demonstrated that AdipoR1 is involved in the regulation of Ca2+ signaling, peroxisome proliferator–activated receptor γ coactivator-1α expression and activation, mitochondrial function and oxidative stress, and glucose and lipid metabolism (17). In liver, AdipoR1 increases ceramidase activity and inhibits gluconeogenesis (11,18). In addition, AdipoR1 overexpression in liver was found to reverse insulin resistance and diabetes in the db/db mouse model of obesity (11).

Despite ample evidence of AdipoR1 role in metabolic regulation, there are scarce and conflicting data concerning its regulation under physiological and pathophysiological conditions. We have recently demonstrated that AdipoR1 protein levels in differentiated human primary skeletal muscle cells and C2C12 myotubes are markedly induced compared with undifferentiated cells. Intriguingly, no significant change was detected in AdipoR1 mRNA levels (19), suggesting that posttranscriptional mechanisms could be involved in AdipoR1 regulation.

Two main posttranscriptional mechanisms affecting levels of expressed proteins are mRNA stability and translational control. These processes are modulated efficiently by both RNA-binding proteins (RBPs) and microRNAs (miRNAs) (2022). RBPs associate with specific sequences within the 5′- or 3′-untranslated region (UTR) of many mRNAs, and this association either increases or decreases mRNA stability or translation depending on the particular mRNA sequence and cellular stimuli (20). miRNAs comprise a large family of small (21–23 nucleotides) noncoding RNAs that inhibit translation or destabilize target mRNAs by binding to their 3′-UTRs with partial base pairing (21). Recent studies have shown that deregulation of miRNAs contributes to the development of obesity-induced insulin resistance (2325).

In the current study, we found that PTB, a RBP mainly known for its role in multiple aspects of mRNA life cycle and function, and miR-221 associate with AdipoR1-3′UTR and cooperatively repress AdipoR1 translation leading to decreased adiponectin signaling. Importantly, this mechanism of regulation is at least partly responsible for the robust induction in AdipoR1 protein levels during myogenesis and the reduction in AdipoR1 protein observed in both liver and muscle during obesity. Thus, we identified a novel posttranscriptional mechanism by which AdipoR1 and its pleiotropic adiponectin-stimulating actions are regulated.

Research Design and Methods

Primer sequences are listed in Supplementary Table 1.

Construction of cDNA Expression Plasmids and Adenoviruses

cDNA fragment of human AdipoR1 was obtained from muscle tissue by RT-PCR, digested with BamHI and HindIII, and cloned into pcDNA 3.1 (Invitrogen) in frame with a Flag coding sequence. Firefly luciferase constructs for AdipoR1-3′ and AdipoR1-5′ UTR have previously been described (19). Mutant AdipoR1-3′UTR luciferase construct was generated by in vitro mutagenesis (Stratagene) and sequenced. pET15b bacterial expression vector expressing gAD (gift from Lily Dong, University of Texas, San Antonio, TX) was transformed into BL21 (DH3) cells and gAD was prepared as previously described (26). The plasmid used for miRNA expression was miR-Vec-221. The miR-Vec retroviral vector contains the genomic region of the pre-miRNA under a strong cytomegalovirus (CMV) promoter (gift from Reuven Agami, the Netherlands Cancer Institute, the Netherlands). The plasmid was sequenced to confirm that the miR-221 miRbase identical sequence is present in its complete form. Flag-tagged PTB was cloned from pcDNA Flag-PTB (gift from Douglas Black, University of California, Los Angeles, Los Angeles, CA) into pAd-Track-CMV plasmid, and adenovirus-expressing Flag-tagged PTB was generated using the pAd-Track-CMV/Ad-Easy adenoviral vector system (Stratagene) according to the manufacturer's instructions.

Cell Cultures, Transfections, and Adenoviral Infections

Mouse primary muscle cells were obtained by enzymatic disaggregation of gastrocnemius muscles from 3-week-old C57BL/6 mice. For separation of myofibers, muscles were incubated with 2 mg/mL collagenese (Sigma) in Dulbecco’s modified Eagle’s medium (DMEM) for 3 h at 37°C. Separated myofibers were isolated and transferred into Matrigel covered plates (BD) and grown for 72 h in BIOAMF-2 medium (Biological Industries) to allow for satellite cell delamination. For enrichment of primary myoblasts, cells were trypsynized and preplated for 1 h, and nonadherent cells were transferred to Matrigel covered plates. Differentiation was induced by incubating an 80% confluent culture in DMEM containing 4% horse serum and 0.04 units/mL human insulin. Primary human skeletal muscle cells (h-SkMcs) were purchased from PromoCell, grown according to manufacturer’s instructions, and differentiated by changing the growth medium to h-SkMc differentiation medium (PromoCell) supplemented for 10 days. HepG2 and C2C12 cells were grown in DMEM supplemented with 10% FBS. Differentiation of C2C12 myoblasts was induced by incubating an 85% confluent culture in DMEM with 2% horse serum. For small interfering RNA (siRNA) experiments, cells were transfected with 100 nmol/L of the following siRNAs: siGENOME SMARTpool PTB-siRNA, siGENOME SMARTpool AdipoR1-siRNA, siGENOME NT-siRNA, miRIDIAN Hairpin inhibitor miR-221 (anti–miR-221), or Hairpin inhibitor negative control (anti–miR-control), using DharmaFECT1 transfection reagent (Dharmacon). For plasmid overexpression experiments, cells were transfected by lipofectamine 2000 (Invitrogen). For adenoviral overexpression studies, C2C12 myotubes were infected 60 h after initiation of differentiation with PTB or green fluorescent protein (GFP)-expressing adenoviruses.

Analysis of De Novo Translation

Nascent AdipoR1 translation was studied by incubating C2C12 myoblasts and myotubes with 1 mCi L-[35S]methionine (Easy Tag Express; NEN/Perkin-Elmer) per 100-mm plate for 30 min. Cells were lysed in radioimmunoprecipitation assay buffer (10 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 1% NP-40; 1 mmol/L EDTA; 0.1% SDS; and 1 mmol/L dithiothreitol), and immunoprecipitation (IP) reactions were carried out in 1 mL TNN buffer (50 mmol/L Tris-HCl, pH 7.5; 250 mmol/L NaCl; 5 mmol/L EDTA; and 0.5% NP-40) for 16 h at 4°C using anti-AdipoR1 (Abcam) or IgG (Sigma) antibodies. After extensive washes in TNN buffer, IP samples were resolved by SDS-PAGE, transferred onto nitrocellulose membrane, and visualized by autoradiography.

RNA Isolation, RT-PCR, and Quantitative PCR Analysis

RNA from frozen tissue or cultured cells was reverse transcribed and quantified with Applied Biosystems Real-Time PCR System and Sybr-green PCR master mix for mRNAs or the TaqMan MicroRNA reverse transcription kit for miRNAs. Gene expression levels were normalized to TATA-binding protein (TBP) or actin mRNAs, 18S rRNA, or U6 snRNA.

IP for RBP Complexes

Association of endogenous PTB and ELAV-like protein 1 (HuR) with endogenous AdipoR1 mRNA and association of PTB with miR-221 were assessed in C2C12 myoblasts and myotubes using a previously described method (27). RNA in IP material was extracted and used in quantitative PCR (qPCR) to detect the presence of mRNAs encoding AdipoR1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), luciferase, miR-133b, and miR-221 using High Capacity cDNA or TaqMan MicroRNA reverse transcription kits (Applied Biosystems).

Reporter Assays

Luciferase reporter assays in HepG2 and C2C12 myoblasts and myotubes were performed using the Dual Luciferase Assay System (Promega) as previously described (19).

Protein Isolation and Western Blotting

Protein extracts from cells and tissues were prepared by solubilization with extraction buffer (19), and samples were resolved by SDS-PAGE and immunoblotted with the following antibodies: AdipoR1 (Abcam), PTB, HuR, myogenin, AdipoR2 (Santa Cruz), phospho-AMPK (Thr172), phospho–acetyl-CoA carboxylase (ACC) (Ser79), total AMPK (t-AMPK) (Cell Signaling), Flag, α-tubulin (Sigma), and total ACC (t-ACC) (Upstate Cell Signaling Solutions).

Animal Studies

C57BL/6 and ob/ob male mice (Harlan Laboratories) were maintained on a 12-h light/dark cycle. For high-fat diet (HFD) experiments, 6-week-old C57BL/6 male mice were maintained on chow or 60% kcal from fat (Research Diets) for 16 weeks. Liver and skeletal muscle tissues were harvested and frozen at −80°C. Glucose and insulin measurements and intraperitoneal glucose tolerance tests were performed as previously described (28). All protocols for animal uses were reviewed and approved by the Animal Care Committee of the Sheba Medical Center and were in accordance with Institutional Animal Care and Use Committee guidelines.

Statistical Analysis

Two-tailed Student t test was used to determine P values. Statistical significance was defined as P < 0.05 and P < 0.01 as indicated.

Results

AdipoR1 Protein in Skeletal Muscle Is Posttranscriptionally Regulated

To study the regulation of AdipoR1 protein in skeletal muscle, we examined protein and mRNA levels of AdipoR1 during myoblast-myotube differentiation in both mouse primary muscle and C2C12 cells. AdipoR1 protein was markedly upregulated in both cell cultures between day 1 and 2 of differentiation, whereas AdipoR1 mRNA did not change during myogenesis (Fig. 1A and B). Additionally, no significant difference in AdipoR1 mRNA stability was observed between myoblasts and myotubes (Supplementary Fig. 1). These findings suggest that at least part of AdipoR1 regulation in muscle is posttranscriptional.

Figure 1

Translational regulation of AdipoR1. A and B: AdipoR1 protein levels are induced during myogenesis. Mouse primary muscle (A) and C2C12 (B) cells were grown in differentiation medium for the times indicated, and total protein and RNA were extracted. Whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. Total RNA was subjected to qPCR analysis with the AdipoR1 gene and normalized to TBP. C: Differentiation promotes AdipoR1 translation. Nascent AdipoR1 production was monitored after a 30-min incubation of C2C12 myoblasts and myotubes with l-[35S]methionine. After IP using either anti-AdipoR1 antibody or control IgG, the incorporation of radiolabeled amino acids into the newly synthesized AdipoR1 protein was assessed by electrophoresis and visualized by autoradiography. D: Luciferase activity of AdipoR1-3′UTR in C2C12 myoblasts and myotubes. Cells were transfected with pGL3 plasmid expressing AdipoR1-3′UTR (LUC-R1-3′UTR) or PGL3 control plasmid (LUC). Firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. Error bars indicate SD. n = 3–5. **P < 0.01.

Given the robust increase in AdipoR1 protein during differentiation, we postulated that AdipoR1 translation is elevated in myogenesis. To test this hypothesis, we monitored the rate of nascent AdipoR1 translation by performing a brief incubation with l-[35S]methionine in C2C12 myoblasts and myotubes. After cell lysis, nascent AdipoR1 was visualized by immunoprecipitation of radiolabeled material using either anti-AdipoR1 or IgG antibodies (as control). This assay revealed a markedly elevated de novo translation of AdipoR1 in C2C12 myotubes compared with myoblasts (Fig. 1C).

Since 3′UTRs are known to play a major role in translational regulation (29), we investigated whether AdipoR1-3′UTR is involved in AdipoR1 translational control by assessing its ability to regulate luciferase activity when expressed downstream of the luciferase gene. Incorporation of AdipoR1-3′UTR into luciferase reporter vector resulted in a significant reduction of luciferase activity in myoblasts but not in myotubes, culminating in an ∼2.5 fold increase in luciferase activity in myotubes compared with myoblasts (Fig. 1D). This result suggests that interaction of cellular factor/s with AdipoR1-3′UTR in undifferentiated cells represses AdipoR1 translation, and this repression is relieved during myogenesis, enabling efficient translation of AdipoR1.

PTB Binds to AdipoR1-3′UTR in Myoblasts but Not Myotubes

RBPs bind to specific cis-acting elements located at the 5′- or 3′UTR (20). Bioinformatics analysis (AREsite and SFmap) identified several putative binding sites for the RBPs HuR and PTB within the AdipoR1-3′UTR sequence. To test whether HuR and PTB bind the endogenous AdipoR1 mRNA, RNA-IP assays were performed. These experiments revealed a similar enrichment of AdipoR1 mRNA in both C2C12 myoblasts and myotubes immunoprecipitated with anti-HuR antibody compared with IgG. However, when we used anti-PTB antibody, a specific and significant enrichment of AdipoR1 mRNA was observed only in C2C12 myoblasts but not myotubes (Fig. 2A). These results indicate that while both PTB and HuR interact with AdipoR1 mRNA in undifferentiated cells, PTB binding to AdipoR1 mRNA is significantly reduced during myogenesis, suggesting that PTB might be involved in AdipoR1 translational inhibition. The differential binding of PTB to AdipoR1 mRNA in myoblasts and myotubes could result from differences in PTB protein levels. Indeed, PTB was significantly reduced during C2C12 and mouse primary muscle cell differentiation (Fig. 2B). Next, we confirmed that PTB binds AdipoR1-3′UTR by assaying chimeric reporters containing the luciferase coding region along with either the 5′UTR or 3′UTR of AdipoR1 (Fig. 2C). After transfection of these constructs into C2C12 myoblasts, we performed RNA-IP assays using anti-PTB or control (IgG) antibodies and tested by qPCR the abundance of LUC, LUC-R1-5′UTR, or LUC-R1-3′UTR mRNAs in IP materials. This analysis (Fig. 2C) revealed that PTB preferentially bound the 3′UTR-containing luciferase chimeric transcript, although a limited binding of PTB to the 5′UTR-containing luciferase chimeric transcript was also observed. Taken together, these findings indicate that PTB binds AdipoR1-3′UTR in undifferentiated muscle cells and that during muscle differentiation PTB levels are reduced and its binding to AdipoR1 mRNA declines.

Figure 2

PTB binds AdipoR1-3′UTR. A: Binding of endogenous PTB and HuR to endogenous AdipoR1 mRNA. Lysates from C2C12 myoblasts or myotubes were used for RNA-IP analysis with PTB, HuR, or IgG (as control) antibodies. RNA was extracted from IP samples, and expression levels of AdipoR1 and GAPDH (control) mRNAs were analyzed by qPCR. Association of AdipoR1 with PTB or HuR is expressed as the relative enrichment compared with the levels of GAPDH mRNA and each mRNA in IgG control samples. B: PTB is downregulated during myogenesis. C2C12 myoblasts or mouse primary muscle cells were grown in differentiation medium for the indicated times. Whole-cell lysates were subjected to Western blot analysis using PTB and α-tubulin antibodies. C: PTB binds AdipoR1-3′UTR in heterologous reporter analysis. C2C12 myoblasts were transfected with constructs expressing luciferase (LUC), luciferase fused to AdipoR1 5′UTR (LUC-R1 5′UTR), and luciferase fused to AdipoR1 3′UTR (LUC-R1 3′UTR) as depicted. Forty-eight hours later, cells were harvested and cytoplasmic lysates were used in IP reactions with either mouse IgG (control) or anti-PTB antibodies. qPCR analysis was used to measure the amount of luciferase or chimeric luciferase mRNAs present in the IP reaction mixtures. Error bars indicate SD. n = 3. **P < 0.01; *P < 0.05.

AdipoR1 Protein Expression Is Regulated by PTB

For functional assessment of PTB’s role in AdipoR1 protein regulation, PTB was depleted in C2C12 myoblasts (which express high PTB and low AdipoR1 protein levels) using a siRNA duplex. After PTB depletion, a robust increase in AdipoR1 protein was detected without any parallel induction in AdipoR1 mRNA (Fig. 3A and B). Importantly, PTB knockdown did not change the proliferative conditions of the cells, as evident by similar cyclin-D1 levels in both control and PTB-depleted cells (Fig. 3A). To test whether PTB knockdown would abrogate AdipoR1 translational inhibition mediated by its 3′UTR (as seen in Fig. 1D), we transfected LUC-R1-3′UTR or LUC plasmids into control or PTB-depleted C2C12 myoblasts and measured luciferase activity. Results (Fig. 3C) demonstrated that luciferase activity of LUC-R1-3′UTR was significantly higher in PTB-depleted cells, thus resembling the induction in luciferase activity of LUC-R1-3′UTR observed during myoblast-myotube differentiation (Fig. 1D).

Figure 3

PTB regulates AdipoR1 expression. C2C12 myoblasts were transfected with siRNA against PTB (si PTB) or control siRNA (si CTRL). Three days posttransfection, total protein and RNA were extracted. A: Whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. B: Total RNA was subjected to qPCR analysis for the indicated genes. C: Luciferase activity of AdipoR1-3′UTR in PTB-depleted C2C12 myoblasts. C2C12 cells were transfected with pGL3 construct expressing AdipoR1-3′UTR (LUC-R1 3′UTR) or pGL3 control plasmid (LUC) and with siRNA against PTB or control siRNA. Two days after transfection, firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. D: C2C12 myotubes were infected with adenoviral vectors expressing Flag-tagged PTB (adPTB) or GFP as control. Two and three days postinfection, whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. Quantification of AdipoR1 protein levels normalized to α-tubulin (% AdipoR1) is shown. E: HepG2 cells were transfected with siRNA against PTB (si PTB) or control siRNA (si CTRL). Three and 6 days posttransfection, protein samples were subjected to Western blot analysis with the indicated antibodies and total RNA was subjected to qPCR analysis for AdipoR1 (normalized to TBP). Error bars indicate SD. n = 3. **P < 0.01.

As shown in Fig. 1A and B, AdipoR1 levels increase dramatically during myogenesis in parallel to a reduction in PTB levels (Fig. 2B). We therefore examined the impact of PTB overexpression on AdipoR1 levels in C2C12 myotubes. After differentiation, myotubes were infected with adenoviral vectors expressing Flag-tagged PTB or GFP as control. Three days postinfection, overexpression of PTB reduced AdipoR1 protein (Fig. 3D) but not mRNA (Supplementary Fig. 2). Importantly, no reduction in myogenin levels was observed indicating that PTB overexpression did not alter differentiation per se. Collectively, these findings demonstrate that PTB inhibits AdipoR1 translation in muscle cells by binding to AdipoR1-3′UTR.

Since AdipoR1 mediates some of adiponectin's pleiotropic effects in liver (10,11,18), we inquired whether PTB regulates AdipoR1 also in hepatocytes. PTB depletion in HepG2 cells (Fig. 3E) significantly increased AdipoR1 protein but not mRNA, suggesting that PTB regulation of AdipoR1 translation is not restricted to skeletal muscle and is most probably a general mechanism to control AdipoR1 levels. Unlike AdipoR1, levels of AdipoR2, which also mediates adiponectin signaling in liver (11), did not change during PTB depletion (Fig. 3E).

Increased AdipoR1 Expression Augments Adiponectin Signaling

AdipoR1 serves as a receptor for globular and full-length adiponectin and mediates increased AMPK activity, fatty acid oxidation, and glucose uptake by adiponectin in muscle (10,1315). Ectopic expression of AdipoR1 in C2C12 myoblasts leads to a significant induction of AMPK phosphorylation (Supplementary Fig. 3), suggesting that increasing AdipoR1 expression in undifferentiated muscle cells enhances adiponectin signaling. To explore the functional significance of PTB regulation of AdipoR1 translation, PTB was knocked down by siRNA in C2C12 myoblasts and phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase (ACC) was examined. PTB depletion dramatically induced AdipoR1 protein levels resulting in increased phosphorylation of AMPK and ACC under both basal and adiponectin-stimulated conditions (Fig. 4). These findings demonstrate that increasing AdipoR1 levels, either by ectopic overexpression or endogenously, by modifying PTB expression, activates adiponectin signaling in skeletal muscle cells.

Figure 4

PTB depletion increases adiponectin signaling in C2C12 myoblasts. C2C12 myoblasts were transfected with siRNA against PTB (siPTB) or control siRNA (siCTRL). Three days posttransfection, cells were serum starved for 6 h and 5 μg/mL gAD was added for 10 min where indicated. Whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. Quantification of AdipoR1, PTB (normalized to α-tubulin), phospho- (p-)AMPK (normalized to t-AMPK), and p-ACC (normalized to t-ACC) protein levels is shown. Error bars indicate SD. n = 3. **P < 0.01; *P < 0.05.

miR-221 Regulates AdipoR1 Protein Expression

Very recently, PTB was implicated in modulating miRNA targeting in mammalian cells (30,31). We therefore investigated the possibility that miRNAs are also involved in AdipoR1 translational regulation. An unbiased bioinformatics search using several databases that predict miRNA targets (TargetScan, microRNA.org, and RNA hybrid) identified miR-221 as a potential miRNA that targets AdipoR1 by binding to its 3′UTR. Notably, miR-221 expression, similarly to PTB, is significantly reduced during muscle cell differentiation (Fig. 5A and ref. 32). For validation that miR-221 regulates AdipoR1 translation, luciferase activity of LUC-R1-3′UTR or LUC was monitored in C2C12 myoblasts transfected with miR-221 antagomir or antagomir control. Overexpression of miR-221 antagomir significantly increased reporter activity of LUC-R1-3′UTR (Fig. 5B), suggesting that miR-221 inhibits AdipoR1 translation by binding to its 3′UTR. We next examined AdipoR1 protein and mRNA levels upon miR-221 overexpression using HepG2 cells. As seen in Fig. 5C, miR-221 overexpression significantly reduced AdipoR1 but not AdipoR2 protein levels (Fig. 5C-1), whereas AdipoR1 mRNA was unaltered (Fig. 5C-2), highlighting an miR-221 role in AdipoR1 translational control. To test whether miR-221 represses AdipoR1 through the binding site identified by bioinformatics analysis, we constructed a luciferase reporter vector fused to AdipoR1-3′UTR with three point mutations in the miR-221 seed region (LUC-R1-3′UTRmut) that disrupts the predicted miR-221 interaction site (Fig. 5D-1). Next, we transfected HepG2 cells expressing miR-221 or control miRNA, with LUC-R1-3′UTR or LUC-R1-3′UTRmut. miR-221 decreased luciferase activity of LUC-R1-3′UTR but had no effect on LUC-R1-3′UTRmut (Fig. 5D-2). Taken together, these findings indicate that miR-221 specifically binds to AdipoR1-3′UTR and decreases AdipoR1 translation.

Figure 5

miRNA-221 regulates AdipoR1 levels. A: miR-221 levels are reduced during myogenesis. Mouse primary muscle cells were grown in differentiation medium for the indicated times, and RNA was extracted. miR-221 levels were measured by qPCR analysis and normalized to U6 snRNA. B: Luciferase activity of AdipoR1-3′UTR in miR-221-depleted C2C12 myoblasts. Cells were transfected with pGL3 construct expressing AdipoR1-3′UTR (LUC-R1 3′UTR) or pGL3 control plasmid (LUC) and with antagomir against miR-221 (anti–miR-221) or anti–miR-control. Seventy-two hours after transfection, firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. C: AdipoR1 levels are reduced upon miR-221 overexpression. HepG2 cells were transfected with vector expressing pre–miR-221 or control vector. Four days posttransfection, whole-cell lysates were subjected to Western blot analysis with the indicated antibodies (C1). Total RNA was subjected to qPCR analysis with the indicated genes (C2). Protein levels of AdipoR1 (normalized to α-tubulin) are shown as the percentage of AdipoR1 levels in control cells. D: Luciferase activity of wild-type or mutant AdipoR1-3′UTR in HepG2 cells overexpressing miR-221. D1: Schematic representation of wild-type (R1-3′UTR) or AdipoR1-3′UTRmut (R1 3′UTRmut) binding to miR-221. D2: HepG2 cells were transfected with pGL3 constructs expressing AdipoR1-3′UTR (LUC-R1 3′UTR) or AdipoR1-3′UTRmut (LUC-R1 3′UTRmut) and with vector expressing pre–miR-221 (miR-221) or control vector (miR-control). Forty-eight hours after transfection, firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. Error bars indicate SD. n = 3. **P < 0.01; *P < 0.05.

PTB and miR-221 Cooperatively Regulate AdipoR1 Protein Expression

To test the notion that PTB regulates AdipoR1 translation in conjunction with miR-221, we investigated whether PTB and miR-221 are both components of the same holo-complex by performing RNA-IP assays. Whole-cell lysates from C2C12 myoblasts or myotubes were immunoprecipitated with anti-PTB or control antibodies, and the presence of miR-221 or miR-133b (an miRNA expressed abundantly in muscle and used as control) was measured by qPCR of IP material. Results (Fig. 6A) demonstrate that PTB complexes are significantly and specifically enriched with miR-221 in myoblasts. As expected, miR-221 was not detected in PTB complexes in myotubes (Fig. 6A), most probably since both PTB and miR-221 are significantly downregulated during myogenesis. To critically examine whether binding of PTB and miR-221 is mediated by AdipoR1 mRNA, we knocked down AdipoR1 in C2C12 myoblasts and assessed the presence of miR-221 in PTB complexes. Results clearly show that while expression levels of both PTB and miR-221 did not change, miR-221 binding to PTB was significantly reduced upon AdipoR1 depletion (Fig. 6B). These findings suggest that PTB, miR-221, and AdipoR1 mRNA are all part of the same complex; moreover, the interaction of PTB with miR-221 is through AdipoR1 mRNA.

Figure 6

Cooperative regulation of PTB and miR-221 on AdipoR1-3′UTR. A: miR-221 binds PTB complexes. Lysates from C2C12 myoblasts or myotubes were used for RNA-IP analysis with PTB or IgG (as control) antibodies. The association of endogenous PTB with miR-221 or miR-133b was tested by qPCR and is expressed as the relative enrichment compared with the level of each miRNA in IgG samples. B: AdipoR1 mRNA mediates miR-221 binding to PTB. C2C12 myoblasts were transfected with siRNA against AdipoR1 (si AdipoR1) or control siRNA (si CTRL). RNA-IP analysis was performed 3 days posttransfection with PTB or IgG antibodies. miR-221 levels in IP material as well as gene expression of AdipoR1, PTB, and miR-221 in total RNA were determined using qPCR analysis. Association of PTB with miR-221 is expressed as the relative enrichment compared with the levels of U6 snRNA and each mRNA in IgG control samples. C: PTB regulates AdipoR1-3′UTR in conjunction with miR-221. C2C12 myoblasts were transfected with pGL3 constructs expressing AdipoR1-3′UTR (LUC-R1 3′UTR), AdipoR1-3′UTRmut (LUC-R1 3′UTRmut), or pGL3 control (LUC) and with siRNA against PTB (si PTB) or control siRNA. Forty-eight hours after transfection, firefly luciferase activities were assessed and normalized to Renilla values as a control for transfection efficiencies. Luciferase expression of siCTRL was set to 1. Error bars indicate SD. n = 3. **P < 0.01; *P < 0.05.

To address whether beyond binding, the cooperative regulation of AdipoR1 by PTB and miR-221 could be functionally demonstrated, we manipulated PTB and miR-221 abilities to bind AdipoR1-3′UTR and examined AdipoR1 translation in C2C12 myoblasts using reporter assays. LUC, LUC-R1-3′UTR, or LUC-R1-3′UTRmut was transfected into PTB depleted or control C2C12 myoblasts, and luciferase activity was monitored. While PTB silencing enhanced luciferase activity of LUC-R1-3′UTR, in keeping with the data described thus far, it did not enhance luciferase activity of LUC-R1-3′UTRmut (Fig. 6C), indicating that miR-221 binding to AdipoR1 mRNA is required for PTB-mediated inhibition of AdipoR1 translation. Overall, these results suggest that PTB and miR-221 coregulate AdipoR1 expression by binding to AdipoR1 mRNA and that PTB inhibits AdipoR1 synthesis by facilitating miR-221 binding to AdipoR1-3′UTR.

Obesity Is Associated With High PTB and miR-221 Levels and Low AdipoR1 Protein Expression

Our results, thus far, indicate that PTB and miR-221 bind AdipoR1-3′UTR and corepress AdipoR1 translation. This suggests that high expression of PTB or miR-221 will result in low protein levels of AdipoR1 and vice versa. Indeed, both PTB and miR-221 levels are lower in gastrocnemius muscle compared with liver, whereas AdipoR1 protein is considerably higher in muscle (Fig. 7A). To examine this novel posttranscriptional regulation in a pathophysiological context, we investigated the levels of AdipoR1 and PTB/miR-221 in muscle and liver in HFD and ob/ob mice, respectively. Importantly, HFD mice were insulin resistant and glucose intolerant, as evidenced by glucose and insulin levels and intraperitoneal glucose tolerance tests (Supplementary Fig. 4). In these obese models, PTB levels were significantly higher while AdipoR1 protein was markedly downregulated (Fig. 7B and C) compared with lean counterparts. Additionally, miR-221 was significantly induced in liver of ob/ob mice and displayed elevated, although not statistically significant, expression in muscle of HFD mice (Fig. 7B and C). Overall, these findings support the notion that AdipoR1 translational control plays a significant role in obesity through modulation of PTB/miR-221 expression in liver and muscle. Notably, AdipoR1 protein was significantly upregulated, whereas expression of PTB and miR-221 was significantly downregulated during differentiation of primary h-SkMcs (Fig. 7D), suggesting that the regulation of PTB and miR-221 on AdipoR1 translation exists also in primary human muscle cells.

Figure 7

PTB, miR-221, and AdipoR1 levels in vivo. Whole-cell lysate and total RNA were extracted from liver, spleen, and skeletal muscle tissues of C57BL/6 mice (A); skeletal muscle of C57BL/6 mice fed for 16 weeks chow (control) or HFD (B); or livers of ob/ob mice or C57BL/6 mice as control (C). PTB and AdipoR1 proteins were determined by Western blot analysis. α-Tubulin was used as a loading control. Quantification of muscle and liver PTB and AdipoR1 immunoblots were performed using ImageJ analysis. miR-221 levels were detected by qPCR analysis and are expressed relative to U6 snRNA levels. D: PTB, miR-221, and AdipoR1 levels in primary human skeletal muscle cells. Whole-cell lysate and total RNA were extracted from h-SkMcs myoblasts and myotubes. AdipoR1, PTB, MHC, and α-tubulin were detected by Western blot analysis, and miR-221 levels were detected by qPCR analysis and are expressed relative to U6 snRNA levels. E: Model proposing a role for PTB and miR-221 in AdipoR1 translational regulation. See the text for details. Error bars indicate SD. n ≥ 3. **P < 0.01; *P < 0.05. MHC, major histocompatibility complex.

Discussion

AdipoR1 has a pivotal role in the pleiotropic actions of adiponectin in vivo, and therefore its gene expression has been studied extensively under various metabolic conditions (7,3337). However, conflicting data about AdipoR1 mRNA expression suggest that transcriptional regulation cannot fully account for AdipoR1 protein expression. Here, we provide the first report that AdipoR1 biosynthesis in both liver and skeletal muscle is regulated at the translational level through binding of the RBP PTB and miR-221 to AdipoR1-3′UTR. Our results indicate that PTB and miR-221 act cooperatively to repress AdipoR1 expression. High levels of these two factors result in significant inhibition of AdipoR1 translation, while reduction in the levels of PTB or miR-221 relieves the translational repression of AdipoR1.

PTB plays critical roles in mRNA metabolism and regulates mRNA splicing, polyadenylation, and mRNA stability (38,39). During muscle differentiation, PTB levels decrease by posttranscriptional mechanisms and allows splicing of muscle-specific transcripts such as NCAM1, ITGA7, and CAPN3 supporting an essential role for PTB in skeletal muscle (40,41). We find here that the decline in PTB levels during myogenesis induces AdipoR1, a protein important for muscle physiology owing to its regulatory role in glucose metabolism and insulin sensitivity. Importantly, the reduction of PTB during muscle differentiation enhances AdipoR1 translation due to decreased binding to AdipoR1-3′UTR, highlighting a novel role for PTB in translation inhibition (Fig. 7E).

Previous studies have implicated RBPs such as HuR, Dnd1, CRD-BP, and PUM1 in modulating miRNA targeting in mammalian cells (42,43). Very recently, PTB has joined this group of RBPs and was shown to be extensively involved in the regulation of miRNA functions (30,31), suppressing or enhancing miRNA targeting by competitive binding on target mRNA or by altering local RNA secondary structure (31). In a similar way, our results suggest that interaction between PTB and the AdipoR1 transcript may promote conformational changes in the secondary structure of the mRNA, thus enabling miR-221 to gain access to its binding site in the 3′UTR of AdipoR1 and thereby enhance repression of AdipoR1 translation (Fig. 7E). In addition to the role of PTB and miRNAs in regulation of mRNA stability (31), our study highlights the interplay between PTB and miRNAs in the control of translation efficiency. Given that posttranscriptional regulation by RBPs and miRNAs targets a specific subset of binding sequences in the 3′UTR of mRNAs, it would be interesting to explore whether additional genes are controlled at the translational level by the cross-talk of miR-221 and PTB.

Inducing AdipoR1 protein levels in C2C12 myoblasts either exogenously, by overexpression, or endogenously, through PTB depletion, resulted in a significant increase in AMPK phosphorylation, one of the primary pathways in adiponectin signaling (Fig. 4 and Supplementary Fig. 3). Indeed, it was shown that the two adiponectin receptors, AdipoR1 and AdipoR2, are capable of homo- and hetero-dimerization, and changes in their cellular ratio/amounts lead to alterations in AMPK phosphorylation (44). Activation of adiponectin signaling was also observed in C2C12 myotubes under depletion of adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), an AdipoR1-binding protein that acts as a negative regulator of adiponectin signaling (45). This study argued that under basal conditions, APPL2 interacts with AdipoR1 and inhibits downstream phosphorylation of AMPK and other signaling molecules activated by AdipoR1, while adiponectin treatment stimulates APPL2 disassociation from AdipoR1, thus leading to its activation. As APPL2 levels are unaltered under PTB depletion (data not shown), a plausible explanation for AdipoR1 activation offered by our study is that increased expression of AdipoR1 results in “free” AdipoR1 molecules, which are not bound to APPL2, thus circumventing adiponectin requirement for AdipoR1 activation and inducing adiponectin signaling. It is important to note that the effects of activation of AdipoR1 may also be mediated by ceramidase activation (18). A recent study demonstrated that AdipoR1 overexpression in 293T cells and in liver significantly enhanced AdipoR1-dependent ceramidase activity, suggesting that increasing AdipoR1 levels can induce ceramide catabolism within the context of adiponectin signaling (18).

Our findings show that muscle and hepatic protein levels of AdipoR1 are significantly reduced in obesity in parallel to induction of PTB and miR-221. In accordance with our findings, earlier studies have demonstrated increased miR-221 expression in liver and adipose tissue of both ob/ob and HFD mice (46,47). Very recently, expression levels of miR-221 were found to positively correlate with BMI in human adipose tissue biopsies (48). Moreover, AdipoR1 was identified as an miR-221 target in adipose tissue (48) and in breast cancer cell lines (49), further supporting our observation that miR-221 is involved in AdipoR1 downregulation in liver and muscle. Interestingly, PTB and miR-221 are differentially expressed in liver and muscle; furthermore, PTB levels were significantly upregulated in both liver and muscle of obese animals, whereas miR-221 expression was significantly induced only in liver, suggesting that the expression of both factors might be tissue and metabolic-condition specific. Overall, the in vivo results in combination with our cell culture studies indicate that alteration in either PTB or miR-221 levels and the cross-talk between these two factors have a significant role in AdipoR1 biosynthesis under physiological and pathophysiological conditions. Nevertheless, since AdipoR1 mRNA levels in muscle were shown to decrease in different models of obesity and diabetes (33,35,37), we cannot exclude that transcriptional regulation also takes place under these conditions.

The identification in the current study of a novel pathway that regulates AdipoR1 translation in muscle and liver could potentially lead to development of drugs that will interfere with PTB/miR-221 binding to AdipoR1 and enhance AdipoR1 synthesis, thereby relieving obesity-associated adiponectin resistance. Moreover, since adiponectin is significantly reduced in obesity and diabetes, our study suggests that increasing AdipoR1 levels could be a logical approach to providing new treatment modality for insulin resistance and the metabolic syndrome.

Article Information

Acknowledgments. The authors acknowledge the critical reading of the manuscript by Assaf Rudich (Ben-Gurion University, Israel).

This work was performed in partial fulfillment of the requirements for the PhD degree for E.B., R.A.-F., and R.G. (Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat-Gan, Israel).

Funding. This work was supported by research grants from the Israeli Association of the Study of Diabetes (to Y.L. and H.K.), the Israel Cancer Association (to H.K.), the Hendrik and Irene Gutwirth Research Scholarships in diabetes (to A.K. and H.K.), the D-Cure Foundation for Diabetes Cure in Israel (to Y.L.), and the Israeli Science Foundation to Yoram Groner, Department of Molecular Genetics, Weizmann Institute of Science. Y.L. was supported by a postdoctoral fellowship from the International Human Frontier Science Program Organization and by a grant from the Israel Ministry of Immigrant Absorption.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Y.L. contributed to the study design, researched and analyzed data, and wrote and edited the manuscript. E.B. and R.A.-F. researched and analyzed data and reviewed the manuscript. R.G. and K.B.-U. researched data. N.S. provided research material, contributed to discussion, and reviewed the manuscript. R.H. contributed to study design and reviewed the manuscript. A.K. contributed to discussion and reviewed the manuscript. H.K. designed the study, participated in results interpretation, and wrote and reviewed the manuscript. H.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

  • Received July 1, 2013.
  • Accepted October 10, 2013.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

References

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  1. Diabetes vol. 63 no. 2 433-445
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