Multiple Signals Induce Endoplasmic Reticulum Stress in Both Primary and Immortalized Chondrocytes Resulting in Loss of Differentiation, Impaired Cell Growth, and Apoptosis

Abstract

The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1β or tumor necrosis factor α induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology. The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1β or tumor necrosis factor α induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology. Cartilage is a connective tissue that serves multiple functions in the developing embryo and in postnatal life. The chondrocyte is the only resident cell found in cartilage and is responsible for both synthesis and turnover of the abundant extracellular matrix (ECM). 1The abbreviations used are: ECM, extracellular matrix; ER, endoplasmic reticulum; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; TN, tunicamycin; TG, thapsigargin; Z, benzyloxycarbonyl; PI, propidium iodide; fmk, fluoromethylketone; IRC, immortalized rat chondrocytes; SNP, sodium nitroprusside. 1The abbreviations used are: ECM, extracellular matrix; ER, endoplasmic reticulum; IL, interleukin; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; TN, tunicamycin; TG, thapsigargin; Z, benzyloxycarbonyl; PI, propidium iodide; fmk, fluoromethylketone; IRC, immortalized rat chondrocytes; SNP, sodium nitroprusside. During development, chondrocytes proliferate and secrete ECM forming the cartilage template of the skeleton (1Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2054) Google Scholar). In the adult, the chondrocytes secrete and maintain ECM that plays a role in growth, mechanical support, and the function of diarthrodial joints between bones (2Olsen B.R. Reginato A.M. Wang W. Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (757) Google Scholar, 3Archer C.W. Francis-West P. Int. J. Biochem. Cell Biol. 2003; 35: 401-404Crossref PubMed Scopus (211) Google Scholar). The chondrocytes reside in an ECM that is neither vascularized nor innervated, therefore nutrient and waste exchange occurs through diffusion under both normal and pathological conditions. The cartilage ECM is composed of collagenous proteins, mainly type II in hyaline cartilage, glycoproteins such as link protein, proteoglycans such as aggrecan, and glycosaminoglycans including chondroitin sulfate, heparin sulfate, and hyaluronic acid (4Eyre D.R. Semin. Arthritis Rheum. 1991; 21: 2-11Crossref PubMed Scopus (104) Google Scholar). The precursors of these macromolecules are synthesized in the endoplasmic reticulum (ER), modified and matured in the Golgi, and secreted and assembled into the ECM (5Vertel B.M. Velasco A. LaFrance S. Walters L. Kaczman-Daniel K. J. Cell Biol. 1989; 109: 1827-1836Crossref PubMed Scopus (35) Google Scholar, 6Luo W. Kuwada T.S. Chandrasekaran L. Zheng J. Tanzer M.L. J. Biol. Chem. 1996; 271: 16447-16450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7McLaughlin S.H. Bulleid N.J. Matrix Biol. 1998; 16: 369-377Crossref PubMed Scopus (97) Google Scholar). A malfunction in this process because of internal or external problems may harm the chondrocyte and/or result in the production of a defective ECM that causes skeletal abnormalities (8Vertel B.M. Walters L.M. Grier B. Maine N. Goetinck P.F. J. Cell Sci. 1993; 104: 939-948Crossref PubMed Google Scholar). For example, pseudoachondroplasia (PASCH) and multiple epiphyseal dysplasia (MED) are autosomal dominant skeletal dysplasias resulting from mutations in the gene coding for cartilage oligomeric protein (COMP), a glycoprotein found in the ECM (9Briggs M.D. Hoffman S.M. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (419) Google Scholar, 10Hecht J.T. Nelson L.D. Crowder E. Wang Y. Elder F.F. Harrison W.R. Francomano C.A. Prange C.K. Lennon G.G. Deere M. Nat. Genet. 1995; 10: 325-329Crossref PubMed Scopus (311) Google Scholar). PSACH and MED patients are characterized by short stature, joint pain, and early onset of osteoarthritis (11Maddox B.K. Keene D.R. Sakai L.Y. Charbonneau N.L. Morris N.P. Ridgway C.C. Boswell B.A. Sussman M.D. Horton W.A. Bachinger H.P. Hecht J.T. J. Biol. Chem. 1997; 272: 30993-30997Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). At the ultrastructural level there are enlarged ER vesicles containing accumulated COMP along with type IX collagen and aggrecan (12Delot E. Brodie S.G. King L.M. Wilcox W.R. Cohn D.H. J. Biol. Chem. 1998; 273: 26692-26697Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13Vranka J. Mokashi A. Keene D.R. Tufa S. Corson G. Sussman M. Horton W.A. Maddox K. Sakai L. Bachinger H.P. Matrix Biol. 2001; 20: 439-450Crossref PubMed Scopus (72) Google Scholar). Another example is Nanomelia, a lethal autosomal recessive defect in chickens resulting from production of defective aggrecan which accumulates in the ER and disrupts normal chondrocyte metabolism (6Luo W. Kuwada T.S. Chandrasekaran L. Zheng J. Tanzer M.L. J. Biol. Chem. 1996; 271: 16447-16450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Vertel B.M. Walters L.M. Grier B. Maine N. Goetinck P.F. J. Cell Sci. 1993; 104: 939-948Crossref PubMed Google Scholar). These studies highlight the importance of the ER-Golgi system for the normal function of chondrocytes. In eukaryotic cells, the ER is a major storage organelle for calcium and site of synthesis and folding of secretory proteins, cell membrane proteins, and lysosomal proteins, prior to their transport to the Golgi for further modification (14Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (586) Google Scholar). Under physiological and pathological conditions, such as nutrient deprivation and oxidative stress, the function of the ER is impaired resulting in “ER stress” (15Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1911) Google Scholar). During ER stress, cells may undergo metabolic arrest and activate specific pathways to survive (16Rao R.V. Ellerby H.M. Bredesen Cell PubMed Scopus Google Scholar). the functions of the ER are may to the cells from the (16Rao R.V. Ellerby H.M. Bredesen Cell PubMed Scopus Google Scholar, M. M. 2003; PubMed Scopus Google Scholar). ER stress plays an important role in cartilage biology. a of such as stress G.R. D.L. 2003; Full Text Full Text PDF PubMed Scopus Google oxidative stress Sci. and mechanical stress R.J. S. Google which may on matrix synthesis J. Arthritis Rheum. 2000; PubMed Scopus Google Scholar, D.R. Google Scholar). In addition, the of cartilage the chondrocytes sensitive to of such as glucose and is the ER stress in chondrocytes or the of ER stress on chondrocyte and matrix gene expression. A report that multiple may induction of ER stress in chondrocytes Y. Tanzer M.L. J. Cell Scopus Google Scholar). this used a immortalized chondrocyte cell and report a regarding the of agents to induce ER stress in this cell studies are to the of of ER stress on chondrocytes and the consequences of ER stress. Here we that a of including glucose induce ER stress markers in primary and immortalized chondrocytes. Further, we demonstrate that ER stress chondrocyte growth, causes down-regulation of cartilage matrix expression at both the protein and mRNA and chondrocyte Cell and rat chondrocytes in as J. G. M. K. Y. Cell PubMed Scopus Google Scholar). These cells have a characterized that is to chondrocytes J. G. M. K. Y. Cell PubMed Scopus Google Scholar). with cells in at a of of chondrocytes from of cartilage was and and with for at in containing and in with at a of of The cells in with or The used thapsigargin acid sodium and Cell Cell was used to the of the cells, to the of the cells in and at or of the into of and and at for Cell was a by and in with as M.D. A. Horton A. Cell Biol. PubMed Scopus Google Scholar). the by and The protein was the with as a of protein was by at for in with of cell protein on and to the cell and system The in for and with the primary was used at rat was used at was used at II was used at was used at used at with the with or at with was an system and as M.D. Horton J. Cell Biochem. 2003; PubMed Scopus (28) Google Scholar). was by DNA was by with to the was the was at for for quantitative real-time PCR as M.D. Horton J. Cell Biochem. 2003; PubMed Scopus (28) Google and synthesized by The are as protein protein PCNA PCNA used at used at and was used as the to have a of to the The quantitative PCR was as M.D. Horton J. Cell Biochem. 2003; PubMed Scopus (28) Google Scholar). the PCR the The was used to the PCR and and expression levels the by B.R. Reginato A.M. Wang W. Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (757) Google Scholar). to of or are Alcian Blue Alcian Blue was as P. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). the was the cell was with PBS, and the in for and at with Alcian Blue in two with acid and two with PBS, the was with with of sodium and the was at and cells in in conditions as with from and to the of the cells in for with glucose withdrawal, tunicamycin, and thapsigargin for and for and with was are as of in DNA was by DNA cells with a and with the DNA was and on a by as S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in at and annexin was to the of the For of collagen II, chondrocytes on with and with for by with for at The and cells with and with for at with PBS, cells with an II for by a at type II was used as with type II for at at The cells under a of levels was as P. K. In Cell Dev. Biol. 1998; PubMed Scopus Google Scholar). of was from and by with of A and of The level was by a at The of the was with a by a of in of was with a of ER in under was that of ER function in chondrocytes by multiple result in a ER stress was the ER stress growth and protein S. M. Cell PubMed Scopus Google Scholar). cells in containing glucose GADD153 for to in glucose deprivation resulted in a induction of GADD153 at which at this to ER stress-inducing is a of that causes in glycoprotein between the ER and Golgi, ER stress and in cell C. P. Curr. Opin. Cell Biol. 2001; 13: PubMed Scopus Google Scholar). is a of the of the ER, which potentially ER stress by ER calcium P. M.D. Nat. Rev. Biol. 2000; PubMed Scopus Google Scholar). Both and thapsigargin resulted in induction of GADD153 in chondrocytes to a that with glucose at immortalized chondrocyte may be for ER is to that an be in primary chondrocytes. rat chondrocytes to the and an of induction of GADD153 was as with the chondrocytes is a which is in the cells to ER stress (16Rao R.V. Ellerby H.M. Bredesen Cell PubMed Scopus Google Scholar). In to the induction of ER stress in we expression. in glucose deprivation both and in chondrocytes. with there was into at In to glucose withdrawal, and both and in chondrocytes such as and have a of on chondrocytes including of cartilage matrix synthesis and of matrix through the production of J. Arthritis Rheum. 2000; PubMed Scopus Google Scholar). In this we the of to induce ER stress, as by GADD153, to agents that perturb ER of chondrocytes with or production cells, there was no induction of in cells with the ER stress induction of GADD153 was with or SNP, and only a minimal was with ER in Matrix in ER is a site of protein synthesis and as as a site of for cartilage matrix by chondrocytes. we that ER stress be by on extracellular matrix cells exposed to glucose withdrawal, and as At and the cell was with Alcian and the was and Under conditions, there was a in In there was no accumulation of under ER stress-inducing conditions with the decreased matrix the chondrocytes exposed to ER stress-inducing conditions changes and accumulated matrix cells with primary rat chondrocytes The Alcian Blue be in to a in cell we studies to at matrix gene and protein expression to chondrocytes. from chondrocytes and primary rat chondrocytes was and used to quantitative real-time PCR with for collagen II, link protein and we to decreased matrix gene expression and by the mRNA coding for proteins was decreased At both the expression of collagen II mRNA was with aggrecan and link protein the primary rat chondrocytes a with to down-regulation of matrix gene expression including the on collagen II The expression level of mRNA no with the at stress in down-regulation of cartilage matrix gene expression in both and primary rat chondrocytes. A and chondrocytes under conditions or exposed to the ER stress-inducing of glucose or or for or was on from for The are as expression of mRNA coding for type II aggrecan, or link protein in chondrocytes. At there is a in mRNA coding for collagen II with with The level of mRNA coding for aggrecan and is at this to a collagen the steady state level of mRNA coding for matrix proteins is by or with chondrocytes. are expression levels of mRNA to the expression level in cells for mRNA for and of steady state coding for collagen II, aggrecan, and was with ER stress-inducing agents in primary rat chondrocytes. there was no in the expression level of the gene coding for with with are the of ER stress in decreased collagen type II chondrocyte in in normal for with and the cells with glucose or for was The are as the of cells with which is at are as of in the a on collagen type II mRNA expression with aggrecan and link protein, we further the of collagen type II under ER stress is a which gene including and aggrecan W. B. Biol. 1997; PubMed Google Scholar). collagen type II was by a collagen type II which collagen type and of site in the site W. B. Biol. 1997; PubMed Google Scholar). ER stress resulted in decreased collagen type II with with glucose and and that type II collagen expression and/or accumulation was decreased in chondrocytes of to ER stress there was no positive in cells by collagen type these that ER stress in an of matrix expression at the level of mRNA that in matrix by chondrocytes. ER in function of the ER is only to the and of protein, to cell we the of induction of ER stress on the growth and of chondrocytes. and primary rat chondrocytes for by and with glucose withdrawal, TN, and chondrocytes an in cell both the and primary chondrocytes to ER stress or with to cell A and cell arrest is responsible for the growth the level of expression of in chondrocytes was was a up-regulation of in chondrocytes under ER stress conditions induced by ER stress In addition, the expression of the was decreased at and under ER stress conditions and decreased PCNA expression ER stress in chondrocytes. expression level was by in chondrocytes under conditions or exposed to ER stress-inducing agents for or was a up-regulation of with ER stress-inducing steady state level of mRNA coding for PCNA was by quantitative real-time PCR in and chondrocytes. ER stress-inducing agents a down-regulation of PCNA are the of ER ER stress result in M. M. 2003; PubMed Scopus Google Scholar). ER stress induce in we a DNA fragmentation cells with glucose withdrawal, TN, or for and was Horton 1998; PubMed Scopus Google that acid in chondrocytes; we used as a positive ER stress-inducing agents the chondrocytes to DNA fragmentation is to with multiple we annexin as a of the DNA with cells, chondrocytes exposed to ER stress annexin V with of these that ER stress in chondrocytes induced by glucose as as agents result in stress in chondrocytes in of annexin cells minimal for annexin V or chondrocytes in the of glucose for abundant with annexin V and minimal with with to and is as an of annexin V and of ER to was that early consequences of ER stress A was to the induction of ER stress chondrocytes of was at with glucose or result in DNA fragmentation at of In addition, the of to the ER stress-inducing agents to the early ER stress from apoptosis, we expression of ER stress markers at two prior to the onset of at there was expression in cells with and by there was an in the level of with of GADD153 was in chondrocytes exposed to glucose and by and at agents induced GADD153 expression with with a The expression of type II collagen was of chondrocyte and a with the ER stress there was of collagen II expression by of with glucose and TG, with a for of collagen II with A was used to the that ER stress resulted in of matrix expression of Here chondrocytes exposed to ER stress-inducing agents in the of a The of the of a DNA at in the of there was up-regulation of GADD153 and of collagen II expression. these that glucose withdrawal, TN, and induce ER stress with a on down-regulation of matrix expression by The stress is an that and cells to to a of and metabolic conditions. ER stress occurs under physiological conditions, and the ER stress plays an role in cell growth and S. M. Cell PubMed Scopus Google Scholar, K. K. A. H. K. R.J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). K. M. Nat. 10: PubMed Scopus (311) Google used a to ER stress in and that ER stress in a of such as and is the role of ER stress in Here we the that a such as decreased glucose induce ER stress in primary rat chondrocytes as as a chondrocyte cell line. The ER stress was to that induced by agents such as and thapsigargin in that both GADD153 and GADD153 is a factor at levels under conditions and is induced by a of that result in ER stress S. M. Cell PubMed Scopus Google Scholar). is an which is induced by ER stress and by of stress H. N. E. J. B.A. J. Nature. 2000; PubMed Scopus Google Scholar). A report that the GADD153 in an immortalized chondrocyte cell and result in Y. Tanzer M.L. J. 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In this we that the of induction of both and is that is of the to from the is which are of normal ER function is with skeletal of collagen in the ER, or an of protein in the ER in ER storage such as multiple epiphyseal dysplasia (MED) (9Briggs M.D. Hoffman S.M. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (419) Google J.T. Nelson L.D. Crowder E. Wang Y. Elder F.F. Harrison W.R. Francomano C.A. Prange C.K. Lennon G.G. Deere M. Nat. Genet. 1995; 10: 325-329Crossref PubMed Scopus (311) Google and type II, M. Nat. 1999; PubMed Scopus Google Scholar, J. M. Google Scholar). Cartilage and cartilage such as osteoarthritis are with G. M. A. J. 2003; PubMed Scopus Google Scholar). In addition, glucose metabolism in the of osteoarthritis Biochem. J. 2003; PubMed Scopus (28) Google Scholar, K. N. M. J. 2001; PubMed Scopus Google Scholar). is to that matrix and chondrocyte cartilage and be to ER stress. In we the of a a specific to ER stress in chondrocytes. In addition, we link the consequences of the ER stress in decreased cartilage matrix decreased chondrocyte growth, and chondrocyte apoptosis, to specific These findings have implications in skeleton development, cartilage cartilage and cartilage In addition, the chondrocyte is an to ER stress to cell for the