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Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways*

  • Weihong Yin
    Affiliations
    Department of Internal Medicine, Section of Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Jong-In Park
    Affiliations
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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  • Richard F. Loeser
    Correspondence
    To whom correspondence should be addressed. Tel.: 336-716-8701; Fax: 336-716-1214
    Affiliations
    Department of Internal Medicine, Section of Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant AG-16697. This work was also supported by an Ellison Medical Foundation/American Federation for Aging Research Postdoctoral Fellowship.
Open AccessPublished:September 17, 2009DOI:https://doi.org/10.1074/jbc.M109.056838
      The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.

      Introduction

      Insulin-like growth factor I (IGF-I)
      The abbreviations used are: IGF-I
      insulin-like growth factor-I
      ROS
      reactive oxygen species
      OA
      osteoarthritis
      IRS
      insulin receptor substrate
      MAPK
      mitogen-activated protein kinase
      PI 3-kinase
      phosphatidylinositol 3-kinase
      ERK
      extracellular signal-regulated kinase
      MEK
      MAPK/ERK kinase
      mTOR
      mammalian target of rapamycin
      p70S6K
      p70 S6 kinase
      MnTBAP
      Mn(III) tetrakis(4-benzoic acid) porphyrin
      NAC
      N-acetyl-l-cysteine
      tBHP
      tert-butylhydroperoxide
      DMEM
      Dulbecco's modified Eagle's medium
      HA
      hemagglutinin
      CA
      constitutively active
      DN
      dominant negative.
      plays a critical role in regulating normal growth and tissue formation during both embryonic and postnatal development through its ability to promote mitogenesis, cell migration, survival, and protein synthesis. Aberrant IGF-I expression or signaling has been implicated in various human diseases, including certain growth disorders (
      • Baker J.
      • Liu J.P.
      • Robertson E.J.
      • Efstratiadis A.
      ), type II diabetes and cardiovascular disease (
      • Ezzat V.A.
      • Duncan E.R.
      • Wheatcroft S.B.
      • Kearney M.T.
      ), osteoarthritis (OA) (
      • Martel-Pelletier J.
      • Di Battista J.A.
      • Lajeunesse D.
      • Pelletier J.P.
      ), and cancer (
      • Samani A.A.
      • Yakar S.
      • LeRoith D.
      • Brodt P.
      ), as well as in cell senescence (
      • Cristofalo V.J.
      • Phillips P.D.
      • Sorger T.
      • Gerhard G.
      ). Importantly, an age-related decline in response to IGF-I stimulation has been noted in musculoskeletal tissues, including bone (
      • D'avis P.Y.
      • Frazier C.R.
      • Shapiro J.R.
      • Fedarko N.S.
      ,
      • Cao J.J.
      • Kurimoto P.
      • Boudignon B.
      • Rosen C.
      • Lima F.
      • Halloran B.P.
      ) and cartilage (
      • Martin J.A.
      • Ellerbroek S.M.
      • Buckwalter J.A.
      ,
      • Loeser R.F.
      • Shanker G.
      • Carlson C.S.
      • Gardin J.F.
      • Shelton B.J.
      • Sonntag W.E.
      ,
      • Messai H.
      • Duchossoy Y.
      • Khatib A.M.
      • Panasyuk A.
      • Mitrovic D.R.
      ), but the underlying mechanisms for this decline are not completely understood.
      IGF-I functions through binding to its cognate cell membrane receptor, IGF-I receptor, which is activated by autophosphorylation and then recruits and activates several signaling intermediates, including members of the insulin-receptor substrate (IRS) family and Shc. Activation of IRS leads downstream to activation of the PI 3-kinase-Akt pathway that activates mammalian target of rapamycin (mTOR), p70S6K, and 4E-BP-1 (eIF-4E-binding protein), key factors regulating mRNA translation and protein synthesis. Activation of Shc leads to activation of the ERK MAPK pathway through Grb2/SOS, Ras, Raf, and the MAPK/ERK kinase (MEK) (
      • Samani A.A.
      • Yakar S.
      • LeRoith D.
      • Brodt P.
      ). MEK1/2 phosphorylates and activates ERK1/2, which, in turn, phosphorylates and activates other kinases and several nuclear factors, such as p90RSK/c-Fos and Elk1. Depending on the cell type and context in which IGF-I stimulation occurs, activation of the PI 3-kinase-Akt pathway most often promotes cell growth, protein synthesis, and/or cell survival, whereas activation of the ERK MAPK pathway promotes cell proliferation and/or gene transcription.
      Aging is a major risk factor for the development of osteoarthritis (OA), the most common joint disease that affects about half of the population over the age of 65 years. OA is characterized by the progressive destruction and loss of the articular cartilage matrix due to unbalanced metabolic activities of the chondrocyte. Compared with those from normal young tissue, chondrocytes isolated from older adult or osteoarthritic cartilage have been found to produce much lower levels of collagen and proteoglycan in response to IGF-I (
      • Martel-Pelletier J.
      • Di Battista J.A.
      • Lajeunesse D.
      • Pelletier J.P.
      ,
      • Martin J.A.
      • Ellerbroek S.M.
      • Buckwalter J.A.
      ,
      • Loeser R.F.
      • Shanker G.
      • Carlson C.S.
      • Gardin J.F.
      • Shelton B.J.
      • Sonntag W.E.
      ). Evidence is accumulating to support a pathogenic role of disrupted redox balance in OA onset and progression, although the mechanisms responsible have not been fully defined (
      • Henrotin Y.E.
      • Bruckner P.
      • Pujol J.P.
      ,
      • Yudoh K.
      • Nguyen T.
      • Nakamura H.
      • Hongo-Masuko K.
      • Kato T.
      • Nishioka K.
      ,
      • Carlo Jr., M.D.
      • Loeser R.F.
      ). There is evidence for increased oxidative stress with aging in human chondrocytes (
      • Carlo Jr., M.D.
      • Loeser R.F.
      ), and chondrocytes from OA tissue have been reported to possess lower levels of anti-oxidant enzymes and increased production of reactive oxygen species (ROS) when compared with normal cartilage (
      • Yudoh K.
      • Nguyen T.
      • Nakamura H.
      • Hongo-Masuko K.
      • Kato T.
      • Nishioka K.
      ,
      • Shah R.
      • Raska Jr., K.
      • Tiku M.L.
      ,
      • Regan E.
      • Flannelly J.
      • Bowler R.
      • Tran K.
      • Nicks M.
      • Carbone B.D.
      • Glueck D.
      • Heijnen H.
      • Mason R.
      • Crapo J.
      ,
      • Aigner T.
      • Fundel K.
      • Saas J.
      • Gebhard P.M.
      • Haag J.
      • Weiss T.
      • Zien A.
      • Obermayr F.
      • Zimmer R.
      • Bartnik E.
      ,
      • Ruiz-Romero C.
      • Calamia V.
      • Mateos J.
      • Carreira V.
      • Martinez-Gomariz M.
      • Fernandez M.
      • Blanco F.J.
      ). Nitrotyrosine, a marker for oxidative damage, has been detected in articular cartilage from older adults and in diseased tissue from people with osteoarthritis, and this correlated with a reduced ability of the articular chondrocytes to produce proteoglycans in response to IGF-I stimulation (
      • Loeser R.F.
      • Carlson C.S.
      • Del Carlo M.
      • Cole A.
      ).
      Because IGF-I utilizes many of the same signaling intermediates as the insulin signaling pathway, shared mechanisms may exist between IGF-I resistance and insulin resistance, the latter of which has been well studied in the context of type II diabetes. An important mechanism contributing to insulin resistance is the inhibition of IRS-1 activation through phosphorylation at several inhibitory serine residues that include Ser-312 and Ser-616 (
      • Aguirre V.
      • Werner E.D.
      • Giraud J.
      • Lee Y.H.
      • Shoelson S.E.
      • White M.F.
      ,
      • Liu Y.F.
      • Herschkovitz A.
      • Boura-Halfon S.
      • Ronen D.
      • Paz K.
      • Leroith D.
      • Zick Y.
      ). Serine phosphorylation of IRS-1 acts to inhibit insulin-mediated tyrosine phosphorylation, the latter of which is required for the ability of IRS-1 to activate the PI 3-kinase pathway. Several mechanisms exist for the induction of IRS-1 serine phosphorylation resulting in insulin resistance, and many of these have inflammation and oxidative stress as common mediators (
      • Wellen K.E.
      • Hotamisligil G.S.
      ).
      To further investigate mechanisms of IGF-I resistance in articular chondrocytes and the potential role of ROS, we compared the activation of key IGF-I signaling intermediates in cells isolated from normal and osteoarthritic cartilage and in normal cells treated with tert-butylhydroperoxide (tBHP). tBHP is a substrate for glutathione peroxidase and, when added to cells, causes oxidative stress by increasing the levels of GSSG at the expense of GSH (
      • Kurz D.J.
      • Decary S.
      • Hong Y.
      • Trivier E.
      • Akhmedov A.
      • Erusalimsky J.D.
      ). The signaling intermediates studied include members of the PI 3-kinase/Akt pathway and the ERK MAPK pathway. Our results show that the balance of PI 3-kinase-Akt and MEK-ERK activity regulates chondrocyte matrix synthesis, and this balance is modulated by oxidative stress that results from elevated levels of ROS.

      DISCUSSION

      This study delineated the roles of two major IGF-I-stimulated signaling pathways, IRS-1-PI 3-kinase-Akt and ERK MAPK, in the regulation of proteoglycan and type II collagen production by human articular chondrocytes and provided evidence that oxidative stress and increased levels of ROS can alter the balance in the activity of these pathways, leading to IGF-I resistance (Fig. 7). Using lentiviral constructs to express CA Akt and CA MEK, we found that Akt activation was sufficient to mediate an increase in proteoglycan synthesis, expression of type II collagen, and type II collagen protein production, whereas activated MEK-ERK was inhibitory for aggrecan expression and proteoglycan synthesis. Although in normal chondrocytes, IGF-I stimulated activation of both the IRS-1-PI 3-kinase-Akt pathway and the MEK-ERK pathway, the phosphorylation of ERK1/2 was more transient relative to Akt. Importantly, the balance in the level of activity of these two pathways was disrupted in chondrocytes isolated from osteoarthritic cartilage such that excessive basal activation of MEK-ERK and an inability of IGF-I to stimulate Akt phosphorylation resulted in an inability of IGF-I to stimulate proteoglycan production. The hypothesis that this imbalance in signaling may result from the excessive levels of ROS that have been observed in OA cartilage (
      • Shah R.
      • Raska Jr., K.
      • Tiku M.L.
      ,
      • Ruiz-Romero C.
      • Calamia V.
      • Mateos J.
      • Carreira V.
      • Martinez-Gomariz M.
      • Fernandez M.
      • Blanco F.J.
      ,
      • Loeser R.F.
      • Carlson C.S.
      • Del Carlo M.
      • Cole A.
      ) was supported by the findings that induction of oxidative stress in normal chondrocytes with tBHP reproduced the changes seen in OA cells, whereas treatment of OA cells with anti-oxidants improved the balance of Akt to ERK phosphorylation and improved the anabolic response of the cells to IGF-I.
      Figure thumbnail gr7
      FIGURE 7Hypothetical model for the effect of oxidative stress on IGF-I regulation of chondrocyte proteoglycan (aggrecan) and collagen II production. In this model, the balance of Akt and ERK1/2 activation determines the final effect of IGF-I on proteoglycan production and collagen expression. In normal chondrocytes, IGF-I activates the IGF-I receptor (IGF-IR), which induces IRS-1 activation through tyrosine phosphorylation and subsequently activates the PI 3-kinase-Akt pathway, which leads downstream to increased aggrecan synthesis and collagen II expression. Increased activation of MEK-ERK signaling, stimulated by oxidative stress and increased ROS levels present in OA cartilage, inhibits aggrecan and collagen gene transcription. Oxidative stress also activates MEK-ERK signaling, which, through inducing IRS-1 serine phosphorylation, negatively regulates IGF-I activation of PI 3-kinase-Akt signaling and proteoglycan synthesis.
      The mechanism for IGF-I resistance in OA cells as well as in normal cells with tBHP induced oxidative stress appeared to be through an inhibition of IRS-1 activation. Studies in chondrocytes from transgenic mice with an IRS-1 deletion have demonstrated that IRS-1 activity is required for IGF-I stimulation of Akt but not ERK1/2 (
      • Shimoaka T.
      • Kamekura S.
      • Chikuda H.
      • Hoshi K.
      • Chung U.I.
      • Akune T.
      • Maruyama Z.
      • Komori T.
      • Matsumoto M.
      • Ogawa W.
      • Terauchi Y.
      • Kadowaki T.
      • Nakamura K.
      • Kawaguchi H.
      ). We noted an increase in phosphorylation of IRS-1 at Ser-312 and Ser-616 in OA cells and in normal cells treated with tBHP. This was associated with a decrease in phosphorylation of IRS-1 at Tyr-612 in response to IGF-I. Activation of IRS-1 requires tyrosine phosphorylation (in particular Tyr-612) (
      • Esposito D.L.
      • Li Y.
      • Cama A.
      • Quon M.J.
      ), whereas serine phosphorylation at a variety of sites has been shown to be inhibitory (
      • Aguirre V.
      • Werner E.D.
      • Giraud J.
      • Lee Y.H.
      • Shoelson S.E.
      • White M.F.
      ,
      • Liu Y.F.
      • Herschkovitz A.
      • Boura-Halfon S.
      • Ronen D.
      • Paz K.
      • Leroith D.
      • Zick Y.
      ). Depending on the specific serine residue, phosphorylation can be mediated through a variety of kinases, including protein kinase C (
      • Liu Y.F.
      • Paz K.
      • Herschkovitz A.
      • Alt A.
      • Tennenbaum T.
      • Sampson S.R.
      • Ohba M.
      • Kuroki T.
      • LeRoith D.
      • Zick Y.
      ,
      • Moeschel K.
      • Beck A.
      • Weigert C.
      • Lammers R.
      • Kalbacher H.
      • Voelter W.
      • Schleicher E.D.
      • Häring H.U.
      • Lehmann R.
      ), IκB kinase (
      • Gao Z.
      • Hwang D.
      • Bataille F.
      • Lefevre M.
      • York D.
      • Quon M.J.
      • Ye J.
      ), mTOR (
      • Gual P.
      • Grémeaux T.
      • Gonzalez T.
      • Le Marchand-Brustel Y.
      • Tanti J.F.
      ), MEK-ERK (
      • Rui L.
      • Aguirre V.
      • Kim J.K.
      • Shulman G.I.
      • Lee A.
      • Corbould A.
      • Dunaif A.
      • White M.F.
      ,
      • Engelman J.A.
      • Berg A.H.
      • Lewis R.Y.
      • Lisanti M.P.
      • Scherer P.E.
      ), and JNK1/2 (
      • Aguirre V.
      • Werner E.D.
      • Giraud J.
      • Lee Y.H.
      • Shoelson S.E.
      • White M.F.
      ,
      • Aguirre V.
      • Uchida T.
      • Yenush L.
      • Davis R.
      • White M.F.
      ).
      Increased activity of MAPKs, including ERK1/2 and JNK1/2, has been reported in OA chondrocytes (
      • Clancy R.
      • Rediske J.
      • Koehne C.
      • Stoyanovsky D.
      • Amin A.
      • Attur M.
      • Iyama K.
      • Abramson S.B.
      ,
      • Fan Z.
      • Söder S.
      • Oehler S.
      • Fundel K.
      • Aigner T.
      ,
      • Boileau C.
      • Martel-Pelletier J.
      • Brunet J.
      • Schrier D.
      • Flory C.
      • Boily M.
      • Pelletier J.P.
      ). We focused the current study on the role of ERK. Increased basal ERK phosphorylation was noted in OA chondrocytes when compared with chondrocytes isolated from normal tissue, and the addition of tBHP to normal cells resulted in ERK phosphorylation. Importantly, a role for ERK in the inhibition of IRS-I-PI 3-kinase-Akt signaling was demonstrated using the MEK1/2 inhibitor U0126 as well as CA MEK expression. MEK1/2 inhibition reduced the level of IRS-I Ser phosphorylation induced by tBHP and increased IGF-I-stimulated IRS-I Tyr and Akt phosphorylation, whereas CA MEK had the opposite effect.
      Our results suggest that increased activity of the ERK MAPK pathway may not only inhibit activation of Akt by IGF-I and thereby inhibit IGF-I-mediated proteoglycan synthesis, but also prolonged ERK activity can inhibit proteoglycan production through down-regulation of aggrecan core protein expression. Aggrecan is the most abundant proteoglycan in articular cartilage and is produced by the addition of glycosaminoglycan chains to the aggrecan core protein. Infection of chondrocytes with lentivirus expressing CA MEK reduced both aggrecan mRNA expression and proteoglycan protein synthesis, as did activation of MEK-ERK using tBHP. Negative regulation of proteoglycan production by activated MEK-ERK has been previously observed during chondrogenesis in embryonic chick limb bud cultures (
      • Bobick B.E.
      • Kulyk W.M.
      ), and inhibition of MEK-ERK has been found to promote chondrocytic differentiation of ATDC5 cells (
      • Phornphutkul C.
      • Wu K.Y.
      • Yang X.
      • Chen Q.
      • Gruppuso P.A.
      ) and aggrecan expression in immortalized rat chondrocytes (
      • Yagi R.
      • McBurney D.
      • Horton Jr., W.E.
      ). These findings are in contrast to a study of mechanically stimulated bovine calf chondrocytes, where inhibition of MEK-ERK either reduced aggrecan expression or had no effect, depending on the stimulation conditions (
      • Fitzgerald J.B.
      • Jin M.
      • Chai D.H.
      • Siparsky P.
      • Fanning P.
      • Grodzinsky A.J.
      ). Thus, the role of MEK-ERK as a positive or negative regulator of chondrocyte aggrecan expression may depend on the stimulus and the contribution of additional signaling pathways that would be stimulus-specific.
      CA MEK also reduced expression of Sox-9, which was accompanied by a modest reduction in type II collagen expression but not collagen protein levels. It should be noted that we measured the total amount of type II collagen protein present in the cell layer rather than new collagen synthesis, so this measurement would include any type II collagen that had already accumulated in the cell layer during the 5 days of culture prior to infection with lentivirus expressing CA MEK.
      Our findings indicate that the relative balance of Akt to ERK activation is important in the ability of IGF-I to stimulate proteoglycan synthesis by adult human articular chondrocytes. The activation of Akt increased proteoglycan synthesis without an increase in aggrecan mRNA levels, suggesting that Akt regulation of proteoglycan synthesis occurs at either the level of protein translation or through regulation of the expression of one or more of the enzymes required for the addition of glycosaminoglycans to the aggrecan core protein or sulfation of the glycosaminoglycan chains. IGF-I activation of the PI 3-kinase-Akt pathway is known to result in an activation of mTOR, a master regulator of mRNA translation and protein synthesis (
      • Hay N.
      • Sonenberg N.
      ), and inhibition of mTOR with rapamycin was previously found to inhibit IGF-I-stimulated proteoglycan synthesis (
      • Starkman B.G.
      • Cravero J.D.
      • Delcarlo M.
      • Loeser R.F.
      ).
      We did observe a significant increase in collagen II and Sox9 mRNA levels in chondrocytes expressing CA Akt, and this was accompanied by an increase in the amount of type II collagen deposited in the matrix. This finding is consistent with the well documented function of Sox-9 as a key transcriptional regulator of the type II procollagen gene (
      • Bi W.
      • Deng J.M.
      • Zhang Z.
      • Behringer R.R.
      • de Crombrugghe B.
      ,
      • Lefebvre V.
      • Huang W.
      • Harley V.R.
      • Goodfellow P.N.
      • de Crombrugghe B.
      ). Because proteoglycan synthesis and collagen II expression are characteristic of the differentiated chondrocyte phenotype, it is possible that Akt promotes and helps in maintaining a differentiated chondrocyte phenotype. This is consistent with a recent study that found that Akt positively regulates chondrocyte maturation and cartilage matrix production during skeletal development (
      • Rokutanda S.
      • Fujita T.
      • Kanatani N.
      • Yoshida C.A.
      • Komori H.
      • Liu W.
      • Mizuno A.
      • Komori T.
      ).
      This study for the first time demonstrated that, in adult human primary chondrocytes, oxidative stress and ERK activation inhibit aggrecan mRNA expression. These findings are consistent with previous studies that demonstrated inhibition of aggrecan expression by H2O2 in juvenile bovine chondrocytes (
      • Martin G.
      • Andriamanalijaona R.
      • Mathy-Hartert M.
      • Henrotin Y.
      • Pujol J.P.
      ) and negative regulation of aggrecan expression by ERK in embryonic chick limb mesenchyme (
      • Bobick B.E.
      • Kulyk W.M.
      ). The ERK inhibition of aggrecan is probably through the activation of its downstream target, c-Fos, which has been found to directly reduce aggrecan expression in the chondrosarcoma cell line HCS 2/8 cells (
      • Tsuji M.
      • Funahashi S.
      • Takigawa M.
      • Seiki M.
      • Fujii K.
      • Yoshida T.
      ). The down-regulation of Sox-9 by CA MEK suggests another possible mechanism because Sox-9, along with l-Sox-5 and Sox-6, has recently been shown to regulate aggrecan expression (
      • Han Y.
      • Lefebvre V.
      ). Further studies will be needed to determine more precisely how ERK negatively regulates aggrecan transcription in primary human chondrocytes.
      Another open question is how oxidative stress activates the MEK-ERK signaling pathway in chondrocytes. ROS have been found to regulate cell signaling at multiple steps in MAPK pathways through activation of a host of kinases as well as through inhibition of various phosphatases (
      • Monteiro H.P.
      • Arai R.J.
      • Travassos L.R.
      ). In rat vascular smooth muscle cells, EGFR transactivation has been shown to be necessary for the ROS activation of ERK1/2 (
      • Meng D.
      • Shi X.
      • Jiang B.H.
      • Fang J.
      ). In our study, pretreatment with an EGFR inhibitor, AG1478, failed to block tBHP activation of ERK1/2 in human chondrocytes.
      W. Yin and R. F. Loeser, unpublished results.
      Further studies are needed to define the mechanism for ROS activation of ERK1/2 in chondrocytes because inhibition of this pathway would be expected to improve the ability to produce proteoglycans in response to IGF-I.
      In summary, we provide evidence that in osteoarthritic chondrocytes and in normal chondrocytes with oxidative stress induced by tBHP, there is an imbalance in IGF-I signaling, resulting in reduced activation of the IRS-1-PI 3-kinase-Akt pathway relative to activation of the MEK-ERK MAPK pathway. Because activation of Akt results in increased expression of Sox9 and type II collagen and increased synthesis of proteoglycans, whereas activation of ERK is inhibitory, this signaling imbalance can have profound effects on chondrocyte matrix production. Additional studies are indicated to determine the mechanism for ROS activation of the MEK-ERK pathway in chondrocytes and how elevated MEK-ERK activity inhibits aggrecan and Sox9 expression. Inhibition of MEK-ERK was found to reduce the loss of cartilage matrix in an animal model of osteoarthritis (
      • Pelletier J.P.
      • Fernandes J.C.
      • Brunet J.
      • Moldovan F.
      • Schrier D.
      • Flory C.
      • Martel-Pelletier J.
      ), suggesting that these findings have in vivo relevance. Another strategy for stimulation of matrix synthesis in OA may be to promote the activity of Akt in chondrocytes and improve the balance of Akt to ERK signaling.

      Acknowledgments

      We thank Yiwen Zhao for technical assistance, Michael Robinson for Akt lentiviral constructs, Natalie Ahn for CA MEK1, and Richard Mulligan for the pHAGE vector. We thank the National Disease Research Interchange (Philadelphia, PA) and the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL) and Drs. Arkady Margolis and Marcello Del Carlo for providing normal donor tissue and Dr. David Martin (Department of Orthopedic Surgery, Wake Forest University School of Medicine) for assistance in obtaining osteoarthritic tissue.

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