Advertisement

Thiazolidinediones Induce Osteocyte Apoptosis by a G Protein-coupled Receptor 40-dependent Mechanism*

  • Aleksandra Mieczkowska
    Affiliations
    From the Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7LD, United Kingdom

    GEROM-LHEA and L'UNAM Université d'Angers, 49933 Angers, France
    Search for articles by this author
  • Michel F. Baslé
    Affiliations
    GEROM-LHEA and L'UNAM Université d'Angers, 49933 Angers, France

    Service Commun d'Imageries et d'Analyses Microscopiques, Institut de Biologie en Santé, L'UNAM Université d'Angers, 49933 Angers, France
    Search for articles by this author
  • Daniel Chappard
    Affiliations
    GEROM-LHEA and L'UNAM Université d'Angers, 49933 Angers, France
    Search for articles by this author
  • Guillaume Mabilleau
    Correspondence
    To whom correspondence should be addressed: GEROM-LHEA, Inst. de Biologie en Santé, IRIS, L'UNAM Université d'Angers, 49933 Angers Cedex 09, France. Tel.: 33-244-688349; Fax: 33-244-688350;
    Affiliations
    From the Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7LD, United Kingdom

    GEROM-LHEA and L'UNAM Université d'Angers, 49933 Angers, France

    Service Commun d'Imageries et d'Analyses Microscopiques, Institut de Biologie en Santé, L'UNAM Université d'Angers, 49933 Angers, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported by grants from the Contrat Région Pays de la Loire: Bioregos2 Program.
    This article contains supplemental Figs. S1 and S2.
      Thiazolidinediones (TZDs) represent an interesting treatment of type 2 diabetes mellitus. However, adverse effects such as heart problems and bone fractures have already been reported. Previously, we reported that pioglitazone and rosiglitazone induce osteocyte apoptosis and sclerostin up-regulation; however, the molecular mechanisms leading to such effects are unknown. In this study, we found that TZDs rapidly activated Erk1/2 and p38. These activations were mediated through Ras proteins and GPR40, a receptor expressed on the surface of osteocytes. Activation of this pathway led only to osteocyte apoptosis but not sclerostin up-regulation. On the other hand, TZDs were capable of activating peroxisome proliferator-activated receptor-γ, and activation of this signaling pathway led to sclerostin up-regulation but not osteocyte apoptosis. This study demonstrates two distinct signaling pathways activated in osteocytes in response to TZDs that could participate in the observed increase in fractures in TZD-treated patients.
      Background: Thiazolidinediones (TZDs) mediate osteocyte apoptosis and sclerostin up-regulation by an unknown mechanism.
      Results: Osteocyte apoptosis is mediated through activation of Erk1/2 and p38, whereas sclerostin up-regulation is through peroxisome proliferator-activated receptor-γ (PPARγ) signaling.
      Conclusion: TZDs signal not exclusively through PPARγ, as thought, but also via a surface receptor called GPR40.
      Significance: Learning how TZDs signal in bone cells is crucial to prevent adverse effects associated with the use of these drugs.

      Introduction

      Thiazolidinediones (TZDs),
      The abbreviations used are: TZD
      thiazolidinedione
      PPARγ
      peroxisome proliferator-activated receptor-γ.
      also known as glitazones, represent a class of pharmaceutical compounds approved for the treatment of type 2 diabetes mellitus. TZDs are peroxisome proliferator-activated receptor-γ (PPARγ) agonists, and as such, upon binding to PPARγ, TZDs promote target gene transcription and protein expression (
      • Kawai M.
      • Rosen C.J.
      PPARγ: a circadian transcription factor in adipogenesis and osteogenesis.
      ). Increased interest for TZDs has emerged with the results of a randomized trial called “A Diabetes Outcome Progression Trial” (ADOPT) demonstrating a durable effect on glycated hemoglobin compared with sulfonylurea or metformin (
      • Kahn S.E.
      • Haffner S.M.
      • Heise M.A.
      • Herman W.H.
      • Holman R.R.
      • Jones N.P.
      • Kravitz B.G.
      • Lachin J.M.
      • O'Neill M.C.
      • Zinman B.
      • Viberti G.
      Glycemic durability of rosiglitazone, metformin, or glyburide monotherapy.
      ). However, adverse effects have been reported with TZD use such as weight gain, fluid retention, and increased risks of congestive heart failure (
      • Kahn S.E.
      • Haffner S.M.
      • Heise M.A.
      • Herman W.H.
      • Holman R.R.
      • Jones N.P.
      • Kravitz B.G.
      • Lachin J.M.
      • O'Neill M.C.
      • Zinman B.
      • Viberti G.
      Glycemic durability of rosiglitazone, metformin, or glyburide monotherapy.
      ,
      • Dormandy J.A.
      • Charbonnel B.
      • Eckland D.J.
      • Erdmann E.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      • Tan M.H.
      • Lefèbvre P.J.
      • Murray G.D.
      • Standl E.
      • Wilcox R.G.
      • Wilhelmsen L.
      • Betteridge J.
      • Birkeland K.
      • Golay A.
      • Heine R.J.
      • Korányi L.
      • Laakso M.
      • Mokán M.
      • Norkus A.
      • Pirags V.
      • Podar T.
      • Scheen A.
      • Scherbaum W.
      • Schernthaner G.
      • Schmitz O.
      • Skrha J.
      • Smith U.
      • Taton J.
      PROactive investigators
      Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomized controlled trial.
      ). Also, an unexplained increased risk of bone fracture has been documented mostly in women, but to date, the causes are unknown (
      • Kahn S.E.
      • Haffner S.M.
      • Heise M.A.
      • Herman W.H.
      • Holman R.R.
      • Jones N.P.
      • Kravitz B.G.
      • Lachin J.M.
      • O'Neill M.C.
      • Zinman B.
      • Viberti G.
      Glycemic durability of rosiglitazone, metformin, or glyburide monotherapy.
      ,
      • Dormandy J.A.
      • Charbonnel B.
      • Eckland D.J.
      • Erdmann E.
      • Massi-Benedetti M.
      • Moules I.K.
      • Skene A.M.
      • Tan M.H.
      • Lefèbvre P.J.
      • Murray G.D.
      • Standl E.
      • Wilcox R.G.
      • Wilhelmsen L.
      • Betteridge J.
      • Birkeland K.
      • Golay A.
      • Heine R.J.
      • Korányi L.
      • Laakso M.
      • Mokán M.
      • Norkus A.
      • Pirags V.
      • Podar T.
      • Scheen A.
      • Scherbaum W.
      • Schernthaner G.
      • Schmitz O.
      • Skrha J.
      • Smith U.
      • Taton J.
      PROactive investigators
      Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomized controlled trial.
      ,
      • Kahn S.E.
      • Zinman B.
      • Lachin J.M.
      • Haffner S.M.
      • Herman W.H.
      • Holman R.R.
      • Kravitz B.G.
      • Yu D.
      • Heise M.A.
      • Aftring R.P.
      • Viberti G.
      Rosiglitazone-associated fractures in type 2 diabetes: an analysis from A Diabetes Outcome Progression Trial (ADOPT).
      ,
      • Meymeh R.H.
      • Wooltorton E.
      Diabetes drug pioglitazone (Actos): risk of fracture.
      ). Several cell types coexist in bone, osteoblasts (bone-forming cells), osteoclasts (bone-resorbing cells), osteocytes (which control bone remodeling), and bone marrow cells, including adipocytes. Adipocytes and osteoblasts come from a common progenitor upon activation of specific transcription factors. Activation of Runx-2 drives progenitors to become osteoblasts, whereas activation of PPARγ results in adipocyte differentiation. As TZDs are PPARγ agonists, it has been postulated that TZDs increase adipocyte differentiation at the expense of osteoblasts in vitro (
      • Lecka-Czernik B.
      • Gubrij I.
      • Moerman E.J.
      • Kajkenova O.
      • Lipschitz D.A.
      • Manolagas S.C.
      • Jilka R.L.
      Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2.
      ,
      • Lecka-Czernik B.
      • Moerman E.J.
      • Grant D.F.
      • Lehmann J.M.
      • Manolagas S.C.
      • Jilka R.L.
      Divergent effects of selective peroxisome proliferator-activated receptor-γ2 ligands on adipocyte versus osteoblast differentiation.
      ). In vivo models showed that TZDs decrease bone formation and increase adiposity in bone marrow, although bone resorption is not affected (
      • Berberoglu Z.
      • Gursoy A.
      • Bayraktar N.
      • Yazici A.C.
      • Bascil Tutuncu N.
      • Guvener Demirag N.
      Rosiglitazone decreases serum bone-specific alkaline phosphatase activity in postmenopausal diabetic women.
      ,
      • Glintborg D.
      • Andersen M.
      • Hagen C.
      • Heickendorff L.
      • Hermann A.P.
      Association of pioglitazone treatment with decreased bone mineral density in obese premenopausal patients with polycystic ovary syndrome: a randomized, placebo-controlled trial.
      ,
      • Grey A.
      • Bolland M.
      • Gamble G.
      • Wattie D.
      • Horne A.
      • Davidson J.
      • Reid I.R.
      The peroxisome proliferator-activated receptor-γ agonist rosiglitazone decreases bone formation and bone mineral density in healthy postmenopausal women: a randomized, controlled trial.
      ,
      • Jennermann C.
      • Triantafillou J.
      • Cowan D.
      • Pennink B.
      • Connolly K.
      • Morris D.
      Effects of thiazolidinediones on bone turnover in the rat.
      ,
      • Rzonca S.O.
      • Suva L.J.
      • Gaddy D.
      • Montague D.C.
      • Lecka-Czernik B.
      Bone is a target for the antidiabetic compound rosiglitazone.
      ).
      The effects of TZDs on osteocytes are poorly understood. In the adult skeleton, osteocytes make up >90–95% of all bone cells compared with 4–6% osteoblasts and 1–2% osteoclasts (
      • Bonewald L.F.
      ). These cells are regularly dispersed throughout the mineralized matrix, connected to each other and to cells on the bone surface through dendritic processes generally radiating toward the bone surface and the blood supply. Osteocytes are a target of drugs affecting bone metabolism such as bisphosphonates because of their connections with blood vessels (
      • Plotkin L.I.
      • Aguirre J.I.
      • Kousteni S.
      • Manolagas S.C.
      • Bellido T.
      Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation.
      ,
      • Plotkin L.I.
      • Lezcano V.
      • Thostenson J.
      • Weinstein R.S.
      • Manolagas S.C.
      • Bellido T.
      Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo.
      ). Osteocytes can conduct and control both bone resorption and bone formation by expressing key mediators such as RANKL and sclerostin (
      • Zhao S.
      • Zhang Y.K.
      • Harris S.
      • Ahuja S.S.
      • Bonewald L.F.
      MLO-Y4 osteocyte-like cells support osteoclast formation and activation.
      ). Recently, we reported that TZDs induce osteocyte apoptosis in a dose-dependent manner (
      • Mabilleau G.
      • Mieczkowska A.
      • Edmonds M.E.
      Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.
      ). Furthermore, we also demonstrated that TZD-treated osteocytes up-regulate the expression of sclerostin, a bone formation inhibitor, whereas RANKL expression is unchanged compared with untreated cells (
      • Mabilleau G.
      • Mieczkowska A.
      • Edmonds M.E.
      Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.
      ). However, the molecular pathways involved in such effects are totally unknown.
      As TZDs are PPARγ agonists, they were thought to signal exclusively through this nuclear receptor. Several recent studies show that TZDs also activate a membrane G protein-coupled receptor called GPR40 (G protein-coupled receptor 40) (
      • Smith N.J.
      • Stoddart L.A.
      • Devine N.M.
      • Jenkins L.
      • Milligan G.
      The action and mode of binding of thiazolidinedione ligands at free fatty acid receptor 1.
      ,
      • Stoddart L.A.
      • Brown A.J.
      • Milligan G.
      Uncovering the pharmacology of the G protein-coupled receptor GPR40: high apparent constitutive activity in guanosine 5′-O-(3-[35S]thio)triphosphate binding studies reflects binding of an endogenous agonist.
      ). GPR40 is a fatty acid receptor activated by long-chain fatty acids. Furthermore, GPR40 is involved in glucose- and fatty acid-induced insulin secretion (
      • Alquier T.
      • Peyot M.L.
      • Latour M.G.
      • Kebede M.
      • Sorensen C.M.
      • Gesta S.
      • Ronald Kahn C.
      • Smith R.D.
      • Jetton T.L.
      • Metz T.O.
      • Prentki M.
      • Poitout V.
      Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets.
      ,
      • Latour M.G.
      • Alquier T.
      • Oseid E.
      • Tremblay C.
      • Jetton T.L.
      • Luo J.
      • Lin D.C.
      • Poitout V.
      GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo.
      ). However, the expression and role of GPR40 in bone are unknown.
      The aim of this study was to investigate the signaling pathways involved in osteocyte apoptosis and sclerostin expression. We found that although TZDs induce sclerostin expression through a PPARγ mechanism, osteocyte apoptosis is mediated via GPR40 and activation of Erk1/2 and p38.

      EXPERIMENTAL PROCEDURES

       Reagents

      Rosiglitazone and troglitazone were purchased from Cayman Chemical (Ann Arbor, MI). Pioglitazone was purchased from Molekula (Shaftesbury, United Kingdom). Signaling inhibitors were purchased from Calbiochem. α-Minimal essential medium, FBS, bovine calf serum, penicillin, and streptomycin were purchased from Lonza (Wokingham, United Kingdom). Antibodies were purchased from Cell Signaling Technology (Danvers, MA) unless indicated otherwise. All other chemicals were purchased from Sigma-Aldrich.

       Animals

      Calvarias (frontal and parietal bones) from 4-week-old female Swiss mice were removed aseptically. The periosteal layers on both side were carefully stripped off with tweezers under α-minimal essential medium, and calvarias were transferred into a collagen-coated T75 cm2 flask prior to culture in α-minimal essential medium supplemented with 5% FBS, 5% bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. At confluency, cells were detached with collagenase and plated as described below. This research was conducted in compliance with appropriate guidelines from the Institutional Animal Care and Use Committee.

       MLO-Y4 Cells

      The murine long bone-derived osteocytic cell line MLO-Y4 was kindly provided by L. Bonewald (University of Missouri-Kansas City, Kansas City, MO). These cells present features of osteocytes (
      • Zhao S.
      • Zhang Y.K.
      • Harris S.
      • Ahuja S.S.
      • Bonewald L.F.
      MLO-Y4 osteocyte-like cells support osteoclast formation and activation.
      ). Cells were cultured in α-minimal essential medium supplemented with 5% FBS, 5% bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were plated at 1 × 104 cells/cm2 on type I collagen-coated plates as described previously (
      • Kato Y.
      • Windle J.J.
      • Koop B.A.
      • Mundy G.R.
      • Bonewald L.F.
      Establishment of an osteocyte-like cell line, MLO-Y4.
      ). Growth arrest was investigated in reducing FBS to 0.5% and bovine calf serum to 0.5%.

       3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Assay

      Cell proliferation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described previously (
      • Dumas A.
      • Gaudin-Audrain C.
      • Mabilleau G.
      • Massin P.
      • Hubert L.
      • Baslé M.F.
      • Chappard D.
      The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility.
      ).

       Osteoclast Cultures

      Bone marrow macrophages from long bones of 4-week-old female Swiss mice were harvested as described previously (
      • Takahashi N.
      • Udagawa N.
      • Tanaka S.
      • Suda T.
      Generating murine osteoclasts from bone marrow.
      ). Osteoclasts were generated using 25 ng/ml recombinant macrophage colony-stimulating factor (R&D Systems), 100 ng/ml recombinant soluble RANKL (PeproTech Ltd., London, United Kingdom), and 10−6 m TZDs. After 7 days of cultures, tartrate-resistant acid phosphatase staining was performed as reported previously (
      • Mabilleau G.
      • Chappard D.
      • Sabokbar A.
      Role of the A20-TRAF6 axis in lipopolysaccharide-mediated osteoclastogenesis.
      ), and multinucleated cells with more than three nuclei were counted as osteoclasts.

       Inhibition of Intracellular Signaling

      To investigate specific signaling pathways, MLO-Y4 cells were cultured in the presence of the signaling inhibitors bisindolylmaleimide I (50 nm), farnesylthiosalicylic acid (20 μm), FR180204 (10 μm), or SB203580 (10 μm) for 1 h prior to the addition of 10−6 m TZDs. These concentrations were selected based on a previous pilot study.
      A. Mieczkowska and G. Mabilleau, unpublished data.
      Sclerostin expression and cell apoptosis were investigated 24 h later as described below.

       Apoptosis Assay

      Apoptosis was determined in TZD-treated cultures as reported previously (
      • Mabilleau G.
      • Mieczkowska A.
      • Edmonds M.E.
      Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.
      ). Briefly, after cell culture, the supernatant containing floating cells was collected and put in previously labeled Eppendorf tubes. Each well was washed with PBS before trypsin was added to detach adherent cells. The mixture containing detached adherent cells was collected and pooled in the Eppendorf tubes containing the cell culture supernatant. Cells were spun at 1500 rpm for 10 min, the supernatant was removed carefully, and cells were incubated with 0.04% trypan blue and transferred into a hemocytometer. Living (clear) and dead (blue) cells were counted under a light microscope, and the percentage of dead cells was determined for each condition as follow: % of dead cells = 100 × (number of dead cells)/(number of dead cells + number of living cells).

       Western Blot Analysis and Immunoblotting

      Cells were cultured in the presence of 10−6 m TZDs for the indicated time periods. Cells were washed with cold PBS, and lysates were made using lysis buffer containing 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 50 mm NaF, 3 mm Na3VO4, protease inhibitor mixture, and 1% Nonidet P-40. Samples were spun at 13,000 rpm for 30 min at 4 °C, the supernatant was collected, and protein concentration was determined with the BCA assay (Thermo Scientific). Samples (20 μg/lane) were run on a 10% acrylamide gel and blotted onto a PVDF membrane. The membranes were washed with TBS and blocked with 5% bovine serum albumin. Samples were incubated overnight with one of the following specific antibodies for Erk1/2: phospho-Erk1/2 (Thr-202/Tyr-204), p38, and phospho-p38 (Thr-180/Tyr-182) (R&D Systems); sclerostin (R&D Systems); PPARγ, phospho-PPARγ (Ser-84), and GPR40 (Santa Cruz Biotechnology); and β-actin (Sigma-Aldrich). Subsequently, membranes were washed with TBS and incubated with the appropriate secondary antibodies coupled to HRP (R&D Systems). Immunoreactive bands were visualized using an ECL kit (Amersham Biosciences). The degree to which the different markers were induced was determined by normalizing the specific signal to that of β-actin using NIH ImageJ software. Control of loading was assessed by Ponceau red staining of the membrane after transfer.

       Silencing

      siGENOME SMARTpool siRNAs (containing a mixture of four siRNAs) targeting murine PPARγ sequences CGAAGAACCAUCCGAUUGA, ACCCAAUGGUUGCUGAUUA, UCACAAUGCCAUCAGGUUU, and CGACAUGAAUUCCUUAAUG; ON-TARGETplus SMARTpool siRNAs targeting murine GPR40 sequences GGAGAAACCUGUUGUGAUU, GGACAAAGUUGCUGAAAUC, GUUCAUAGUUUGAGCGUUA, and GAUAUGAUGUAGAGUUUGA; and nonspecific control siRNA duplexes were purchased from Thermo Scientific. Cells (5 × 103 cells/cm2) were plated in either 6-wells plates or 25-cm2 flasks coated with type I collagen as described above and cultured in the presence of 10−6 m TZDs. After 24 h, cells were washed twice with Opti-MEM I (Invitrogen) and preincubated with a mixture of 100 nm siRNA, Oligofectamine (Invitrogen), and Opti-MEM I. Cells were exposed to this transfection mixture for 16 h before being returned to normal culture medium. Forty-eight hours after transfection, TZDs (10−6 m) were added to the cultures. Knockdown efficiency was assessed by Western blotting.

       Generation of Reactive Oxygen Species

      MLO-Y4 cells were plated at a density of 1 × 104 cells/cm2 and cultured for up to 60 min in the presence of TZDs. At the end of the incubation period, intracellular levels of reactive oxygen species were determining using dichlorofluorescein diacetate as described previously (
      • Mabilleau G.
      • Moreau M.F.
      • Filmon R.
      • Baslé M.F.
      • Chappard D.
      Biodegradability of poly(2-hydroxyethyl methacrylate) in the presence of the J774.2 macrophage cell line.
      ), and fluorescence was read with a M2 microplate reader (Molecular Devices) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. As a positive control, MLO-Y4 cells were incubated in the presence of 100 nm phorbol 12-myristate 13-acetate.

       Transmission Electron Microscopy

      MLO-Y4 cells were fixed in 3.7% paraformaldehyde in Sorensen's buffer. The cells were then dehydrated in a graded series of ethanol and embedded in Lowicryl K4M. Ultrathin sections were cut, and immunodetection of GPR40 and Bax (R&D Systems) were done using a secondary antibody complexed with 10-nm gold beads.

       Statistical Analysis

      Statistical analysis was performed with Systat® statistical software release 11.0 (Systat Software, Inc., San Jose, CA). Results are expressed as means ± S.E. The non-parametric Kruskall-Wallis test was used to compare the differences between the groups. When significant differences were observed, data were subjected to the Mann-Whitney U test. Differences at p < 0.05 were considered significant. Experiments were repeated at least four times.

      RESULTS

       Rapid Activation of Erk1/2 and p38 Is PPARγ-independent

      As represented Fig. 1, within 15 min of incubation with TZDs, a rapid and massive phosphorylation of Erk1/2 and p38 could be noted in osteocytes. This activation lasted for up to 60 min. On the other hand, no phosphorylation of Akt or JNK was recorded in the presence of TZDs (data not shown). As these activations were rapid, we hypothesized that they did not require PPARγ. To test this hypothesis, we performed silencing of this nuclear receptor. Interestingly, after a 15-min treatment with pioglitazone or rosiglitazone, although expression of PPARγ was reduced by 90% after 72 h compared with cells treated with scrambled siRNA, activation of Erk1/2 and p38 was unchanged. This seems to indicate that these two signaling pathways were activated independently of PPARγ. Several previous studies reported a role for TZDs in inducing oxidative stress in cells, leading to activation of MAPK independently of PPARγ. However, no increase in reactive oxygen species generation was recorded in the presence of TZDs (Fig. 1C).
      Figure thumbnail gr1
      FIGURE 1Pioglitazone, rosiglitazone, and troglitazone induce Erk1/2 and p38 phosphorylation via a PPARγ-independent mechanism. A, TZDs at a concentration of 10−6 m induced a rapid activation of Erk1/2 and p38 in MLO-Y4 cells. Black bars, pioglitazone (PIO); white bars, rosiglitazone (ROSI); gray bars, troglitazone (TRO). B, Erk1/2 and p38 activation was investigated in siRNA-treated cultures. Silencing of PPARγ did not affect the pattern of Erk1/2 and p38 activation. sc, scrambled siRNA. C, MLO-Y4 cells treated with phorbol 12-myristate 13-acetate (PMA) significantly increased the production of reactive oxygen species, whereas TZDs failed to induce such response. **, p < 0.01 versus control cultures.

       Activation of Erk1/2 and p38 Is Ras-dependent

      As PKC and Ras are two targets of TZDs in other tissues, we decided to investigate their role in Erk1/2 and p38 activation. The use of bisindolylmaleimide I, a specific inhibitor of PKC, did not reduce the activation of Erk1/2 or p38 in response to pioglitazone or rosiglitazone (Fig. 2A). On the other hand, the use of farnesylthiosalicylic acid, a specific inhibitor of Ras proteins, hampered activation of Erk1/2 and p38 in pioglitazone- or rosiglitazone-treated cells (Fig. 2B). These results suggest that activation of Erk1/2 and p38 is dependent on proteins from the Ras family.
      Figure thumbnail gr2
      FIGURE 2Ras but not PKC is required for Erk1/2 and p38 activation in TZD-treated cultures. Bisindolylmaleimide I (A), a specific inhibitor of PKC, and farnesylthiosalicylic acid (B), a specific inhibitor of Ras, were used in the culture prior to the addition of 10−6 m pioglitazone (PIO) or rosiglitazone (ROSI). Phosphorylation of Erk1/2 and p38 was assessed after 0, 15, 30, and 60 min. **, p < 0.01 versus untreated cells.

       Sclerostin Up-regulation Is PPARγ-dependent, whereas MLO-Y4 Apoptosis Is p38-dependent

      As it seems that TZDs have PPARγ-independent mechanisms, we wanted to determine whether osteocyte apoptosis and sclerostin up-regulation are under PPARγ control (Fig. 3). In the presence of TZDs, we observed an increase in osteocyte apoptosis. Silencing of PPARγ did not modify the pattern of osteocyte apoptosis. These results suggest that TZDs induce osteocyte apoptosis through a PPARγ-independent mechanism (Fig. 3A). On the other hand, sclerostin up-regulation was dramatically decreased by 71 and 54% in pioglitazone- and rosiglitazone-treated cultures, respectively, in the absence of PPARγ (Fig. 3B). Furthermore, in the promoter of the murine sclerostin gene, we found a putative sequence for PPARγ binding at −1832 (data not shown), reinforcing the idea that sclerostin expression might be under the control of PPARγ. Rapidly after activation by TZDs, PPARγ was phosphorylated at Ser-84 (supplemental Fig. S1A). The phosphorylation remained even in the presence of FR180204 and SB203580 (supplemental Fig. S1B). We next investigated whether osteocyte apoptosis and sclerostin up-regulation, in response to TZDs, are mediated by activation of Erk1/2 and/or p38 (Fig. 4). In the presence of 10 μm FR180204, a specific Erk1/2 inhibitor, osteocyte apoptosis was slightly increased by 13 and 10% in pioglitazone- and rosiglitazone-treated cultures, respectively (Fig. 4A). On the other hand, in the presence of 10 μm SB203580, a specific p38 inhibitor, osteocyte apoptosis was significantly decreased by 69 and 63% in pioglitazone- and rosiglitazone-treated cultures, respectively. As Erk1/2 and p38 are known modulators of cell growth, MLO-Y4 cells were cultured in reduced serum conditions. Arresting cell growth did not affect the pattern of osteocyte death, but the presence of SB203580 significantly lowered the amount of dead osteocytes in low serum conditions (supplemental Fig. S2), suggesting that TZDs induce osteocyte death, independently of cell growth, via a p38-dependent mechanism. We also looked at sclerostin expression, and we found that the presence of FR180204 or SB203580 did not significantly affect the pattern of sclerostin expression in response to TZD stimulation.
      Figure thumbnail gr3
      FIGURE 3Sclerostin expression but not osteocyte apoptosis is mediated through PPARγ. A, MLO-Y4 cells were pretreated with scrambled (white bars) or PPARγ (gray bars) siRNA prior to the addition of TZDs, and osteocyte apoptosis was investigated. Untreated cells (black bars) served as controls. B, sclerostin expression was assessed in untreated cells (− and black bars), cells transfected with scrambled siRNA (sc and white bars), and cells transfected with PPARγ siRNA (ppar-γ and gray bars). **, p < 0.01 versus cells transfected with scrambled siRNA. PIO, pioglitazone; ROSI, rosiglitazone.
      Figure thumbnail gr4
      FIGURE 4Osteocyte apoptosis but not sclerostin expression is dependent on Erk1/2 and p38 activation. A, MLO-Y4 cells were pretreated with 10 μm FR180204 (white bars), a specific inhibitor of Erk1/2, or 10 μm SB203580 (gray bars), a specific inhibitor of p38, prior to the addition of 10−6 m TZDs to the culture. Untreated cells served as controls (black bars). B, sclerostin expression was assessed in untreated cells (− and black bars), FR180204-treated cells (FR and white bars), and SB203580-treated cells (SB and gray bars). **, p < 0.01 versus untreated cells. PIO, pioglitazone; ROSI, rosiglitazone.

       MLO-Y4 Apoptosis Is Mediated through GPR40

      In other cell systems, it has been reported that TZDs activate a cell surface receptor called GPR40. We investigated whether osteocytes express GPR40. Indeed, by Western blotting, we found that GPR40 was abundantly expressed in osteocytes (Fig. 5A) but also in human osteoblasts and primary human osteoclasts. Furthermore, immunogold labeling revealed that GPR40, in osteocytes, was localized at the cell membrane (Fig. 5B). To investigate whether GPR40 could be a target for TZDs in osteocytes, we performed silencing experiments. After 72 h, GPR40 expression was significantly reduced by 92% (Fig. 6A). GPR40 silencing resulted in a decreased activation of Erk1/2 and p38 in pioglitazone- and rosiglitazone-treated cultures, although this decrease was more marked for p38 (Fig. 6B). In response to pioglitazone and rosiglitazone, osteocyte apoptosis was significantly decreased by 66 and 70%, respectively, in cells in which GPR40 was silenced (Fig. 6C), confirming that this cell surface receptor is responsible for p38 activation and osteocyte apoptosis. On the other hand, sclerostin expression was unchanged in GPR40-silenced cultures in response to TZDs (Fig. 6D). Moreover, we investigated the intracytoplasmic localization of Bax in TZD-treated cells (Fig. 6E). TZDs triggered a relocalization of Bax from the cytoplasm to the outer membrane of the mitochondria. Silencing of GPR40 or SB203580 treatment reversed the relocalization of Bax.
      Figure thumbnail gr5
      FIGURE 5GPR40 is expressed on the surface of osteocytes. A, detection of GPR40 by Western blotting in protein extracts of human osteoblasts (Ob), murine osteocytes (Ocy), and human primary osteoclasts (Oc). B, low magnification of MLO-Y4 cells (panel a). Immunogold labeling of GPR40 revealed its presence only on the cytoplasmic membrane (panel b). GPR40 was not found in the nuclear membrane (panel c), Golgi apparatus (panel d), mitochondria (panel e), or rough endoplasmic reticulum (panel f). Arrows indicate the localization of gold beads.
      Figure thumbnail gr6
      FIGURE 6Osteocyte apoptosis but not sclerostin expression is GPR40-dependent. A, efficiency of GPR40 silencing. B, silencing of GPR40 resulted in decrease activation of Erk1/2 and p38. White bars, cells transfected with scrambled siRNA (sc); gray bars, cells transfected with GPR40 siRNA. **, p < 0.01 versus cells transfected with scrambled siRNA. C, osteocyte apoptosis was determined in response to TZDs in untreated cells (black bars), cells transfected with scrambled siRNA (white bars), and cells transfected with GPR40 siRNA (gray bars). D, GPR40 silencing did not affect the pattern of expression of sclerostin. White bars, cells transfected with scrambled siRNA; gray bars, cells transfected with GPR40 siRNA. E, intracytoplasmic localization of Bax in untreated cells (panel a), pioglitazone-treated cells (panel b), rosiglitazone (ROSI)-treated cells (panel c), pioglitazone (PIO)-treated cells transfected with scrambled siRNA (panel d), and pioglitazone-treated cells transfected with GPR40 siRNA and pioglitazone- and SB203580-treated cells (panel f).

       TZDs Induce Cell Death and Sclerostin Up-regulation in Primary Osteoblasts

      To ascertain whether the above findings were restricted to the MLO-Y4 cell line or could be extended to bone-derived osteoblasts, we investigated p38 activation, cell death, and sclerostin expression in response to TZDs in osteoblasts obtained from calvarias of young mice (Fig. 7). After pioglitazone treatment, p38 was rapidly activated in primary osteoblasts (Fig. 7A). Interestingly, TZD induced a significant augmentation of cell death in osteoblast cultures by 60 and 72% in pioglitazone- and rosiglitazone-treated cultures, respectively (Fig. 7B). This increase in cell death was mediated, as for MLO-Y4 cells, through a GPR40/p38-mediated mechanism as evidenced by the reduction in cell death in the presence of siRNA targeting GPR40 or SB203580 (Fig. 7, B and C). On the other hand, PPARγ silencing did not affect cell death in TZD-treated cultures (Fig. 7D). Similar to what was observed with MLO-Y4 cells, TZDs significantly up-regulated sclerostin expression. On the other hand, sclerostin expression was not affected by GPR40 silencing (data not shown), but as for MLO-Y4 cells, sclerostin expression was significantly reduced by 84% in cultures in which PPARγ was silenced.
      Figure thumbnail gr7
      FIGURE 7TZD treatment results also in primary osteoblast death and sclerostin up-regulation. A, p38 was activated rapidly after pioglitazone treatment in primary osteoblasts. B, cell death was assessed in primary osteoblasts cultured with 10−6 m TZDs (black bars). Cell death was also determined in osteoblast cultures transfected with either scrambled siRNA (white bars) or GPR40 siRNA (gray bars). **, p < 0.01 versus untreated cells; ##, p < 0.01 versus scrambled siRNA. PIO, pioglitazone; ROSI, rosiglitazone. C, cell death mediated by TZDs was abolished in cells pretreated with SB203580. **, p < 0.01 versus non-pretreated cells. D, silencing of PPARγ did not affect cell death. E, sclerostin expression was significantly augmented in pioglitazone-treated cells but reduced in the absence of PPARγ in pioglitazone-treated cells. ##, p < 0.01 versus scrambled siRNA (sc).

       TZDs Decrease Osteoclast Formation through a GPR40/p38-mediated Mechanism

      As GPR40 was expressed also in osteoclasts, we investigated the role of TZD in osteoclast physiology. In contrast to what was reported for primary osteoblasts and MLO-Y4 cells, TZDs did not increase the death of osteoclast precursors (Fig. 8). However, although cell death was not affected, treatment of osteoclast precursor cultures with TZDs resulted in a dose-dependent decrease in osteoclast numbers. We then postulated that the same GPR40/p38-mediated mechanism might be responsible for this reduction in osteoclast numbers, and indeed, silencing of GPR40 or use of SB203580 significantly increased the number of osteoclast as evidenced in Fig. 8 (C and D).
      Figure thumbnail gr8
      FIGURE 8TZDs modulates osteoclastogenesis through a GPR40/p38-dependent mechanism. A, bone marrow macrophages were cultured in the presence of TZDs for 24 h prior to cell death assessment. PIO, pioglitazone; ROSI, rosiglitazone. B, bone marrow macrophages were cultured in the presence of 25 ng/ml macrophage colony-stimulating factor, 100 ng/ml RANKL, and various concentrations of pioglitazone prior to osteoclast counting. **, p < 0.01 versus previous pioglitazone concentration. C, GPR40 silencing restored osteoclastogenesis. Bone marrow macrophages were pretreated with either scrambled siRNA (white bars) or GPR40 siRNA (gray bars) prior to the addition of 10−6 m TZDs to the culture. Black bars represent untreated cultures. **, p < 0.01 versus cells transfected with scrambled siRNA. D, inhibition of p38 restored osteoclastogenesis. Bone marrow macrophages were pretreated with 10 μm SB203580 (gray bars) prior to the addition of 10−6 m TZDs to the culture. Untreated cells served as controls (black bars). **, p < 0.01 versus cells not treated with SB203580.

      DISCUSSION

      TZDs represent an interesting class of drugs used in the treatment of type 2 diabetes mellitus. However, several adverse effects, including low bone mass and bone fracture, have been reported in patients treated with these molecules. Previously, we reported that TZDs induce sclerostin expression and osteocyte apoptosis (
      • Mabilleau G.
      • Mieczkowska A.
      • Edmonds M.E.
      Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.
      ). However, little was known about the molecular mechanism leading to these two events and especially whether they were the result of distinct molecular pathways or linked. In this study, our results suggest that although TZDs induce sclerostin expression through activation of PPARγ, osteocyte apoptosis is mediated through a different signaling pathway involving GPR40, proteins from the Ras superfamily, and activation of Erk1/2 and p38.
      TZDs have previously been described as agonists of PPARγ. The ligand binding affinity order for PPARγ is rosiglitazone > pioglitazone > troglitazone (
      • Mabilleau G.
      • Chappard D.
      • Baslé M.F.
      Cellular and molecular effects of thiazolidinediones on bone cells: a review.
      ). However, although TZDs mediate osteocyte apoptosis, the ranking order for TZD potency in doing this is pioglitazone > troglitazone > rosiglitazone (
      • Mabilleau G.
      • Chappard D.
      • Baslé M.F.
      Cellular and molecular effects of thiazolidinediones on bone cells: a review.
      ). This finding is in contradiction to the ligand binding affinities. In this study, we have demonstrated that silencing of PPARγ did not affect the pattern of osteocyte apoptosis, whereas silencing of GPR40 significantly decreased osteocyte apoptosis. Furthermore, blockade of p38, which is activated within 15 min in osteocytes, kinetically appears unlikely to reflect actions toward PPARγ. Taken together, these results indicate that although TZDs have been described as strong PPARγ agonists, some effects are mediated through different intracellular signaling pathways. On the other hand, the order of potency regarding sclerostin expression seems to follow the ligand binding affinities and is markedly reduced when PPARγ is silenced, suggesting a PPARγ-dependent mechanism. This idea is further reinforced by the fact that a putative PPARγ-responsive element could be identified in the promoter of the Sost gene. The molecular mechanism leading to PPARγ activation by TZDs is still unclear. PPARγ activity may be modulated by several post-translational modifications, including phosphorylation, sumoylation, ubiquitination, nitration, and intracellular compartmentalization (see review in Ref.
      • Luconi M.
      • Cantini G.
      • Serio M.
      Peroxisome proliferator-activated receptor-γ (PPARγ): is the genomic activity the only answer?.
      ). Some of our results suggest an increase in Ser-84 phosphorylation in response to TZDs; however, this phosphorylation seems to be independent of Erk1/2 and p38 activation, as specific inhibitors such as FR180204 and SB203580 did not modify the pattern of Ser-84 phosphorylation. Further studies are clearly required to fully understand the mechanism behind PPARγ activation and sclerostin up-regulation.
      Although TZDs signal undoubtedly through PPARγ, several other signaling pathways have been evidenced (
      • Smith N.J.
      • Stoddart L.A.
      • Devine N.M.
      • Jenkins L.
      • Milligan G.
      The action and mode of binding of thiazolidinedione ligands at free fatty acid receptor 1.
      ,
      • Colca J.R.
      • McDonald W.G.
      • Waldon D.J.
      • Leone J.W.
      • Lull J.M.
      • Bannow C.A.
      • Lund E.T.
      • Mathews W.R.
      Identification of a novel mitochondrial protein (“mitoNEET”) cross-linked specifically by a thiazolidinedione photoprobe.
      ,
      • Dewar B.J.
      • Gardner O.S.
      • Chen C.S.
      • Earp H.S.
      • Samet J.M.
      • Graves L.M.
      Capacitative calcium entry contributes to the differential transactivation of the epidermal growth factor receptor in response to thiazolidinediones.
      ,
      • Fryer L.G.
      • Parbu-Patel A.
      • Carling D.
      The antidiabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways.
      ,
      • Gardner O.S.
      • Dewar B.J.
      • Earp H.S.
      • Samet J.M.
      • Graves L.M.
      Dependence of peroxisome proliferator-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation.
      ,
      • Gardner O.S.
      • Dewar B.J.
      • Graves L.M.
      Activation of mitogen-activated protein kinases by peroxisome proliferator-activated receptor ligands: an example of nongenomic signaling.
      ,
      • Takeda K.
      • Ichiki T.
      • Tokunou T.
      • Iino N.
      • Takeshita A.
      15-Deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones activate the MEK/ERK pathway through phosphatidylinositol 3-kinase in vascular smooth muscle cells.
      ). Among all of these pathways, similarities in intracellular targets have been evidenced with transactivation of the EGF receptor. As such, in liver cells, transactivation of this receptor (
      • Dewar B.J.
      • Gardner O.S.
      • Chen C.S.
      • Earp H.S.
      • Samet J.M.
      • Graves L.M.
      Capacitative calcium entry contributes to the differential transactivation of the epidermal growth factor receptor in response to thiazolidinediones.
      ,
      • Gardner O.S.
      • Dewar B.J.
      • Earp H.S.
      • Samet J.M.
      • Graves L.M.
      Dependence of peroxisome proliferator-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation.
      ,
      • Gardner O.S.
      • Dewar B.J.
      • Graves L.M.
      Activation of mitogen-activated protein kinases by peroxisome proliferator-activated receptor ligands: an example of nongenomic signaling.
      ) leads to activation of Erk and p38. This pathway involves the rapid generation of reactive oxygen species to activate Src and then the EGF receptor. However, in our study, we demonstrated that, in osteocytes, TZDs did not generate oxidative stress, and it is unlikely that this pathway is at the origin of osteocyte apoptosis. A strong argument to support the role of GPR40 in osteocyte apoptosis is that MAPK signaling and osteocyte apoptosis are strongly inhibited in the presence of GPR40 silencing. Furthermore, several recent studies reported the involvement of GPR40 in PPARγ-independent response to TZDs in other cell types (
      • Gras D.
      • Chanez P.
      • Urbach V.
      • Vachier I.
      • Godard P.
      • Bonnans C.
      Thiazolidinediones induce proliferation of human bronchial epithelial cells through the GPR40 receptor.
      ,
      • Wu P.
      • Yang L.
      • Shen X.
      The relationship between GPR40 and lipotoxicity of the pancreatic β-cells as well as the effect of pioglitazone.
      ). Smith et al. (
      • Smith N.J.
      • Stoddart L.A.
      • Devine N.M.
      • Jenkins L.
      • Milligan G.
      The action and mode of binding of thiazolidinedione ligands at free fatty acid receptor 1.
      ) recently reported the mode of binding of TZDs to GPR40 and demonstrated, upon binding, a rapid activation of Gαq/Gα11, resulting in activation of Erk1/2 and p38. Our study suggests similar results, as TZDs induced Erk1/2 and p38 activation upon interaction with GPR40. Our study also seems to suggest that Ras proteins are intermediates between GPR40 and MAPK activation. However, whether this event requires Gαq/Gα11 and augmentation in intracellular calcium remains unknown.
      To our knowledge, this is the first report of the expression of GPR40, the free fatty acid receptor 1, on the surface of osteocytes. Cornish et al. (
      • Cornish J.
      • MacGibbon A.
      • Lin J.M.
      • Watson M.
      • Callon K.E.
      • Tong P.C.
      • Dunford J.E.
      • van der Does Y.
      • Williams G.A.
      • Grey A.B.
      • Naot D.
      • Reid I.R.
      Modulation of osteoclastogenesis by fatty acids.
      ) previously reported the expression of GPR40 in murine osteoclast precursor cells but not in murine osteoblasts. Cornish et al. demonstrated that the use of GW9508, a GPR40/GPR120 agonist, resulted in decreased osteoclastogenesis. Our results in osteoclast cultures also support a role of GPR40 in reducing osteoclastogenesis. A growing body of evidence suggests that PPARγ is necessary for osteoclastogenesis (
      • Wan Y.
      • Chong L.W.
      • Evans R.M.
      PPARγ regulates osteoclastogenesis in mice.
      ,
      • Wei W.
      • Wang X.
      • Yang M.
      • Smith L.C.
      • Dechow P.C.
      • Sonoda J.
      • Evans R.M.
      • Wan Y.
      PGC1β mediates PPARγ activation of osteoclastogenesis and rosiglitazone-induced bone loss.
      ); however, this is intriguing, as TZDs have been shown to decrease osteoclast formation and bone resorption (
      • Chan B.Y.
      • Gartland A.
      • Wilson P.J.
      • Buckley K.A.
      • Dillon J.P.
      • Fraser W.D.
      • Gallagher J.A.
      PPAR agonists modulate human osteoclast formation and activity in vitro.
      ,
      • Mbalaviele G.
      • Abu-Amer Y.
      • Meng A.
      • Jaiswal R.
      • Beck S.
      • Pittenger M.F.
      • Thiede M.A.
      • Marshak D.R.
      Activation of peroxisome proliferator-activated receptor-γ pathway inhibits osteoclast differentiation.
      ,
      • Okazaki R.
      • Toriumi M.
      • Fukumoto S.
      • Miyamoto M.
      • Fujita T.
      • Tanaka K.
      • Takeuchi Y.
      Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro.
      ). One explanation could be that, in osteoclasts, the observed effect associated with the use of TZDs is mediated through GPR40, and as such, further studies on the impact of GPR40 activation in osteoclasts are needed.
      As a conclusion, Fig. 9 summarizes the effects of TZDs on osteocytes. Rapidly after treatment with TZDs, phosphorylation of Erk1/2 and p38 occurs through the involvement of GPR40, expressed on the cytoplasmic membrane, and Ras. This signaling pathway results in recruitment of Bax to the outer membrane of the mitochondria and induction of osteocyte apoptosis. In parallel, TZDs cross the cytoplasmic membrane and activate PPARγ. In return, PPARγ induces the expression of sclerostin within 24 h.
      Figure thumbnail gr9
      FIGURE 9Schematic representation of TZD action in osteocytes. TZDs activate two distinct signaling pathways. TZDs bind GPR40 and rapidly activate Erk1/2 and p38 through a Ras-dependent mechanism. This pathway leads to recruitment of Bax to the outer membrane of the mitochondria and osteocyte apoptosis. TZDs also bind to PPARγ, and this results in activation of the sclerostin gene (SOST) and sclerostin up-regulation.

      Supplementary Material

      REFERENCES

        • Kawai M.
        • Rosen C.J.
        PPARγ: a circadian transcription factor in adipogenesis and osteogenesis.
        Nat. Rev. Endocrinol. 2010; 6: 629-636
        • Kahn S.E.
        • Haffner S.M.
        • Heise M.A.
        • Herman W.H.
        • Holman R.R.
        • Jones N.P.
        • Kravitz B.G.
        • Lachin J.M.
        • O'Neill M.C.
        • Zinman B.
        • Viberti G.
        Glycemic durability of rosiglitazone, metformin, or glyburide monotherapy.
        N. Engl. J. Med. 2006; 355: 2427-2443
        • Dormandy J.A.
        • Charbonnel B.
        • Eckland D.J.
        • Erdmann E.
        • Massi-Benedetti M.
        • Moules I.K.
        • Skene A.M.
        • Tan M.H.
        • Lefèbvre P.J.
        • Murray G.D.
        • Standl E.
        • Wilcox R.G.
        • Wilhelmsen L.
        • Betteridge J.
        • Birkeland K.
        • Golay A.
        • Heine R.J.
        • Korányi L.
        • Laakso M.
        • Mokán M.
        • Norkus A.
        • Pirags V.
        • Podar T.
        • Scheen A.
        • Scherbaum W.
        • Schernthaner G.
        • Schmitz O.
        • Skrha J.
        • Smith U.
        • Taton J.
        • PROactive investigators
        Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomized controlled trial.
        Lancet. 2005; 366: 1279-1289
        • Kahn S.E.
        • Zinman B.
        • Lachin J.M.
        • Haffner S.M.
        • Herman W.H.
        • Holman R.R.
        • Kravitz B.G.
        • Yu D.
        • Heise M.A.
        • Aftring R.P.
        • Viberti G.
        Rosiglitazone-associated fractures in type 2 diabetes: an analysis from A Diabetes Outcome Progression Trial (ADOPT).
        Diabetes Care. 2008; 31: 845-851
        • Meymeh R.H.
        • Wooltorton E.
        Diabetes drug pioglitazone (Actos): risk of fracture.
        CMAJ. 2007; 177: 723-724
        • Lecka-Czernik B.
        • Gubrij I.
        • Moerman E.J.
        • Kajkenova O.
        • Lipschitz D.A.
        • Manolagas S.C.
        • Jilka R.L.
        Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2.
        J. Cell. Biochem. 1999; 74: 357-371
        • Lecka-Czernik B.
        • Moerman E.J.
        • Grant D.F.
        • Lehmann J.M.
        • Manolagas S.C.
        • Jilka R.L.
        Divergent effects of selective peroxisome proliferator-activated receptor-γ2 ligands on adipocyte versus osteoblast differentiation.
        Endocrinology. 2002; 143: 2376-2384
        • Berberoglu Z.
        • Gursoy A.
        • Bayraktar N.
        • Yazici A.C.
        • Bascil Tutuncu N.
        • Guvener Demirag N.
        Rosiglitazone decreases serum bone-specific alkaline phosphatase activity in postmenopausal diabetic women.
        J. Clin. Endocrinol. Metab. 2007; 92: 3523-3530
        • Glintborg D.
        • Andersen M.
        • Hagen C.
        • Heickendorff L.
        • Hermann A.P.
        Association of pioglitazone treatment with decreased bone mineral density in obese premenopausal patients with polycystic ovary syndrome: a randomized, placebo-controlled trial.
        J. Clin. Endocrinol. Metab. 2008; 93: 1696-1701
        • Grey A.
        • Bolland M.
        • Gamble G.
        • Wattie D.
        • Horne A.
        • Davidson J.
        • Reid I.R.
        The peroxisome proliferator-activated receptor-γ agonist rosiglitazone decreases bone formation and bone mineral density in healthy postmenopausal women: a randomized, controlled trial.
        J. Clin. Endocrinol. Metab. 2007; 92: 1305-1310
        • Jennermann C.
        • Triantafillou J.
        • Cowan D.
        • Pennink B.
        • Connolly K.
        • Morris D.
        Effects of thiazolidinediones on bone turnover in the rat.
        J. Bone Miner. Res. 1995; 10: S241
        • Rzonca S.O.
        • Suva L.J.
        • Gaddy D.
        • Montague D.C.
        • Lecka-Czernik B.
        Bone is a target for the antidiabetic compound rosiglitazone.
        Endocrinology. 2004; 145: 401-406
        • Bonewald L.F.
        Rosen C.J. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7th Ed. American Society for Bone and Mineral Research, Washington, D.C.2008: 22-27
        • Plotkin L.I.
        • Aguirre J.I.
        • Kousteni S.
        • Manolagas S.C.
        • Bellido T.
        Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of extracellular signal-regulated kinase activation.
        J. Biol. Chem. 2005; 280: 7317-7325
        • Plotkin L.I.
        • Lezcano V.
        • Thostenson J.
        • Weinstein R.S.
        • Manolagas S.C.
        • Bellido T.
        Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo.
        J. Bone Miner. Res. 2008; 23: 1712-1721
        • Zhao S.
        • Zhang Y.K.
        • Harris S.
        • Ahuja S.S.
        • Bonewald L.F.
        MLO-Y4 osteocyte-like cells support osteoclast formation and activation.
        J. Bone Miner. Res. 2002; 17: 2068-2079
        • Mabilleau G.
        • Mieczkowska A.
        • Edmonds M.E.
        Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.
        Diabet. Med. 2010; 27: 925-932
        • Smith N.J.
        • Stoddart L.A.
        • Devine N.M.
        • Jenkins L.
        • Milligan G.
        The action and mode of binding of thiazolidinedione ligands at free fatty acid receptor 1.
        J. Biol. Chem. 2009; 284: 17527-17539
        • Stoddart L.A.
        • Brown A.J.
        • Milligan G.
        Uncovering the pharmacology of the G protein-coupled receptor GPR40: high apparent constitutive activity in guanosine 5′-O-(3-[35S]thio)triphosphate binding studies reflects binding of an endogenous agonist.
        Mol. Pharmacol. 2007; 71: 994-1005
        • Alquier T.
        • Peyot M.L.
        • Latour M.G.
        • Kebede M.
        • Sorensen C.M.
        • Gesta S.
        • Ronald Kahn C.
        • Smith R.D.
        • Jetton T.L.
        • Metz T.O.
        • Prentki M.
        • Poitout V.
        Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets.
        Diabetes. 2009; 58: 2607-2615
        • Latour M.G.
        • Alquier T.
        • Oseid E.
        • Tremblay C.
        • Jetton T.L.
        • Luo J.
        • Lin D.C.
        • Poitout V.
        GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo.
        Diabetes. 2007; 56: 1087-1094
        • Kato Y.
        • Windle J.J.
        • Koop B.A.
        • Mundy G.R.
        • Bonewald L.F.
        Establishment of an osteocyte-like cell line, MLO-Y4.
        J. Bone Miner. Res. 1997; 12: 2014-2023
        • Dumas A.
        • Gaudin-Audrain C.
        • Mabilleau G.
        • Massin P.
        • Hubert L.
        • Baslé M.F.
        • Chappard D.
        The influence of processes for the purification of human bone allografts on the matrix surface and cytocompatibility.
        Biomaterials. 2006; 27: 4204-4211
        • Takahashi N.
        • Udagawa N.
        • Tanaka S.
        • Suda T.
        Generating murine osteoclasts from bone marrow.
        Methods Mol. Med. 2003; 80: 129-144
        • Mabilleau G.
        • Chappard D.
        • Sabokbar A.
        Role of the A20-TRAF6 axis in lipopolysaccharide-mediated osteoclastogenesis.
        J. Biol. Chem. 2011; 286: 3242-3249
        • Mabilleau G.
        • Moreau M.F.
        • Filmon R.
        • Baslé M.F.
        • Chappard D.
        Biodegradability of poly(2-hydroxyethyl methacrylate) in the presence of the J774.2 macrophage cell line.
        Biomaterials. 2004; 25: 5155-5162
        • Mabilleau G.
        • Chappard D.
        • Baslé M.F.
        Cellular and molecular effects of thiazolidinediones on bone cells: a review.
        Int. J. Biochem. Mol. Biol. 2011; 2: 240-246
        • Luconi M.
        • Cantini G.
        • Serio M.
        Peroxisome proliferator-activated receptor-γ (PPARγ): is the genomic activity the only answer?.
        Steroids. 2010; 75: 585-594
        • Colca J.R.
        • McDonald W.G.
        • Waldon D.J.
        • Leone J.W.
        • Lull J.M.
        • Bannow C.A.
        • Lund E.T.
        • Mathews W.R.
        Identification of a novel mitochondrial protein (“mitoNEET”) cross-linked specifically by a thiazolidinedione photoprobe.
        Am. J. Physiol. Endocrinol. Metab. 2004; 286: E252-E260
        • Dewar B.J.
        • Gardner O.S.
        • Chen C.S.
        • Earp H.S.
        • Samet J.M.
        • Graves L.M.
        Capacitative calcium entry contributes to the differential transactivation of the epidermal growth factor receptor in response to thiazolidinediones.
        Mol. Pharmacol. 2007; 72: 1146-1156
        • Fryer L.G.
        • Parbu-Patel A.
        • Carling D.
        The antidiabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways.
        J. Biol. Chem. 2002; 277: 25226-25232
        • Gardner O.S.
        • Dewar B.J.
        • Earp H.S.
        • Samet J.M.
        • Graves L.M.
        Dependence of peroxisome proliferator-activated receptor ligand-induced mitogen-activated protein kinase signaling on epidermal growth factor receptor transactivation.
        J. Biol. Chem. 2003; 278: 46261-46269
        • Gardner O.S.
        • Dewar B.J.
        • Graves L.M.
        Activation of mitogen-activated protein kinases by peroxisome proliferator-activated receptor ligands: an example of nongenomic signaling.
        Mol. Pharmacol. 2005; 68: 933-941
        • Takeda K.
        • Ichiki T.
        • Tokunou T.
        • Iino N.
        • Takeshita A.
        15-Deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones activate the MEK/ERK pathway through phosphatidylinositol 3-kinase in vascular smooth muscle cells.
        J. Biol. Chem. 2001; 276: 48950-48955
        • Gras D.
        • Chanez P.
        • Urbach V.
        • Vachier I.
        • Godard P.
        • Bonnans C.
        Thiazolidinediones induce proliferation of human bronchial epithelial cells through the GPR40 receptor.
        Am. J. Physiol. Lung Cell. Mol. Physiol. 2009; 296: L970-L978
        • Wu P.
        • Yang L.
        • Shen X.
        The relationship between GPR40 and lipotoxicity of the pancreatic β-cells as well as the effect of pioglitazone.
        Biochem. Biophys. Res. Commun. 2010; 403: 36-39
        • Cornish J.
        • MacGibbon A.
        • Lin J.M.
        • Watson M.
        • Callon K.E.
        • Tong P.C.
        • Dunford J.E.
        • van der Does Y.
        • Williams G.A.
        • Grey A.B.
        • Naot D.
        • Reid I.R.
        Modulation of osteoclastogenesis by fatty acids.
        Endocrinology. 2008; 149: 5688-5695
        • Wan Y.
        • Chong L.W.
        • Evans R.M.
        PPARγ regulates osteoclastogenesis in mice.
        Nat. Med. 2007; 13: 1496-1503
        • Wei W.
        • Wang X.
        • Yang M.
        • Smith L.C.
        • Dechow P.C.
        • Sonoda J.
        • Evans R.M.
        • Wan Y.
        PGC1β mediates PPARγ activation of osteoclastogenesis and rosiglitazone-induced bone loss.
        Cell Metab. 2010; 11: 503-516
        • Chan B.Y.
        • Gartland A.
        • Wilson P.J.
        • Buckley K.A.
        • Dillon J.P.
        • Fraser W.D.
        • Gallagher J.A.
        PPAR agonists modulate human osteoclast formation and activity in vitro.
        Bone. 2007; 40: 149-159
        • Mbalaviele G.
        • Abu-Amer Y.
        • Meng A.
        • Jaiswal R.
        • Beck S.
        • Pittenger M.F.
        • Thiede M.A.
        • Marshak D.R.
        Activation of peroxisome proliferator-activated receptor-γ pathway inhibits osteoclast differentiation.
        J. Biol. Chem. 2000; 275: 14388-14393
        • Okazaki R.
        • Toriumi M.
        • Fukumoto S.
        • Miyamoto M.
        • Fujita T.
        • Tanaka K.
        • Takeuchi Y.
        Thiazolidinediones inhibit osteoclast-like cell formation and bone resorption in vitro.
        Endocrinology. 1999; 140: 5060-5065