From the
Veterans Affairs Medical Center, University of California San Diego, La Jolla, California 92161,
Molecular Cardiology Unit, Victor Chang Cardiac Research Institute, School of Biochemistry and Molecular Genetics, University of New South Wales, 2010 New South Wales, Australia
Received for publication, January 30, 2003
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ABSTRACT |
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INTRODUCTION |
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In contrast to chondrocytes in growth plate cartilage, chondrocytes in normal articular cartilage remain largely in a resting state and do not undergo terminal differentiation or mineralize their matrix (7). But in OA, foci of articular chondrocyte hypertrophy develop, typically near sites of cartilage surface lesions (8, 9). Chondrocyte hypertrophy in OA may function partly to modulate matrix remodeling and repair (10) and partly to promote calcification, as hypertrophic chondrocytes are commonly co-localized with deposits of hydroxyapatite and calcium pyrophosphate dihydrate crystals in the disease (11). Significantly, deposits of hydroxyapatite and calcium pyrophosphate dihydrate crystals crystals can stimulate intra-articular inflammation and further damage to cartilage in OA (12, 13).
One of the shared features of hypertrophic chondrocytes in growth plate and articular cartilages is up-regulated expression of two transglutaminase (TG) isoenzymes (14, 15). These are factor XIIIA (FXIIIA), a distinct tissue homodimeric form of the circulating heterotetrameric coagulation factor XIII, and TG2 (also termed tissue TG and Gh), which is unique among the human TG isoenzymes in being a dual function TG and GTPase/ATPase (16). TGs catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues to a primary amino group (EC 2.3.2.13
[EC]
). TGs thereby can modify the matrix through effects including protein cross-linking and stabilization (16), and OA cartilage contains a variety of potential TG substrates including several collagen subtypes, fibronectin, and the mineralization-regulatory protein osteopontin (17). Furthermore, TG transamidation catalytic activity has been shown to increase in both an OA severity-dependent and age-dependant manner in joint cartilages (18, 19). We recently observed that certain mediators implicated in OA, including IL-1 (20) stimulates chondrocyte matrix calcification (19). IL-1
also induces TG transamidation catalytic activity in cultured chondrocytes (19). Furthermore, selective "gain-of-function" of either chondrocyte TG2 or FXIIIA TG transamidation activity via transfection was associated with potent up-regulation of the capacity of chondrocytes to calcify their matrix (19).
TG2 was recently observed to regulate vascular smooth muscle cell differentiation (21). In mononuclear phagocytes and fibroblasts, TG2 regulates adhesion and migration (22). Thus, we directly tested the role of TG2 in IL-1-induced calcification by chondrocytes. In addition, we tested the hypothesis that TG2 is not simply a marker of chondrocyte hypertrophy but also a direct mediator of the development of chondrocyte hypertrophic differentiation. To directly probe TG2 functions in chondrocyte differentiation and calcification we have taken advantage of the recent generation of TG2 knockout mice, which are phenotypically normal (23, 24). To specifically assess for direct TG2 involvement in articular chondrocyte hypertrophy, we studied the effects on primary chondrocytes of CNP (25) and of the vitamin A-derived metabolite all-trans retinoic acid (ATRA) (26, 27). CNP is expressed in developing long bones, and the stimulatory effects of CNP on chondrocyte proliferation and hypertrophy are essential for normal growth plate development (25). ATRA promotes maturation to hypertrophy and calcification by chick sternal chondrocytes in vitro (26) in a manner that requires the transcription factor Cbfa1 (27). In addition, hypervitaminosis A and toxicity of certain other retinoids is associated with accelerated terminal differentiation of endochondral chondrocytes and premature epiphyseal closure in vivo (28, 29). Moreover, retinoid treatment of cultured chondrocytes is known to increase expression and activity of TGs (30) in vitro.
Chondrocyte hypertrophic differentiation in both the growth plate and in OA articular cartilage has been heretofore linked closely with a state in which matrix calcification is up-regulated (2, 4, 8). However, it has not been clarified if there is an obligate linkage of up-regulated calcification with chondrocyte hypertrophic differentiation. In this study, we demonstrate, using IL-1 and CNP-stimulated cells, that chondrocyte hypertrophy and up-regulated matrix calcification are dissociable states. We also establish TG2 to be an essential mediator of IL-1
-induced calcification, as well as ATRA-induced Cbfa1 expression, hypertrophic differentiation, and calcification in articular chondrocytes. In contradistinction, we establish that CNP-induced Cbfa1 expression and hypertrophic differentiation do not require TG2 in articular chondrocytes.
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EXPERIMENTAL PROCEDURES |
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TG2 Null MiceWe established a breeding colony of TG2 (+/+) and TG2 (/) mice as previously described (23). The C57BL6/129SVJ heterozygote TG2 (/) mice were bred to generate homozygous TG2 (/) and congenic wild-type founders. Each set of founders was used for only 5 generations to avoid breeding artifacts.
Cell Isolation, Culture Conditions, and Assessment of Matrix CalcificationPrimary articular chondrocytes from normal bovine knees (Animal Technologies, Tyler, TX) were isolated after dissection by collagenase digestion of the tibial plateau and femoral condyle articular cartilage (58). Primary mouse articular chondrocytes were isolated by dissection of the tibial plateaus and femoral condyles of the TG2 wild-type and knockout mice at two months of age. The articular cartilage was carefully peeled off with a scalpel under a dissecting microscope to avoid disruption of the subchondral bone. The cartilage was subsequently digested with 2 mg/ml clostridial collagenase at 37 °C for 2 h and then plated in monolayer culture. Subconfluent chondrocytes were shown to express type II collagen in > 95% of the cells by immunocytochemistry. Approximately 2500 primary chondrocytes were obtained initially from a pair of knee joints from each two-month-old mouse. Mouse chondrocytes were allowed to proliferate for 5 days, which yielded 10,000 cells per pair of knees from an individual mouse. For the described experiments, knees from 30 mice of each genotype were harvested, and the chondrocytes of each genotype were pooled upon isolation and plated at 70% confluency.
Primary chondrocytes were cultured in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal calf serum, 1% glutamine, 100 units/ml penicillin, 50 g/ml Streptomycin (Omega Scientific, Tarzana, CA) and maintained at 37 °C in the presence of 5% CO2 for 7 days prior to the initiation of each experiment. Functional studies on chondrocytes were performed in either Medium A (Dulbecco's modified Eagle's medium high glucose supplemented with 1% fetal calf serum, 1% glutamine, 100 units/ml penicillin, 50 g/ml streptomycin, 1 mM sodium phosphate, and 50 g/ml of ascorbic acid) or Medium B (which had the same composition as Medium A except for a lack of sodium phosphate), unless otherwise indicated.
To quantify matrix calcification by the primary articular chondrocytes, we used a previously described Alizarin Red S binding assay, which was further validated in each experiment by direct visual observation of Alizarin Red S staining in each plate (5). Here, aliquots of mouse chondrocytes (1 x 103 cells/well) were plated in individual wells of 96-well plates in a volume of 0.2 ml of Medium A.
SDS-PAGE/Western Blotting and RT-PCRFor SDS-PAGE/Western blotting, conditioned media was collected at the designated time points and concentrated with cold tricholoracetic acid (final concentration of 15%) for 15 min on ice. Cell lysates and protein pellets from the concentrated media were resuspended in 4% SDS, 0.2 M Tris, pH 6.8, and 40% glycerol, and the protein was determined with the bicinchoninic acid protein assay (Pierce). Aliquots of 0.01 mg protein from each sample were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (31). Anti-MMP-13 (Chemicon, Temecula, CA), anti-type X collagen (Calbiochem, San Diego, CA), anti-FXIIIA (Calbiochem), anti-TG2 (Upstate Biotechnology, Lake Placid, NY), and anit-tubulin were used at a 1:1000 dilution in Western blotting (31).
Total RNA was extracted with Trizol, and RT-PCR was performed as previously described (31). Primers that amplified the ribosomal gene L30 were used as a loading control. We used previously described primers to amplify L30 (31), CD38 (32), TG5 (33) and mouse Cbfa1 (34). TG1 sense (5'-TCAGATGCTGGAGGTGACAG-3') and TG1 antisense (5'-CCCAGTCTTCCTGTCTGAGC-3') primers amplified a 171-bp product (positions 25422712 in the published sequence). TG2 sense (5'-TGCTCCTATTGGCCTGTACC-3') and TG2 antisense (5'-CCAAAGTTCCAAGGCACACT-3') primers amplified a 222-bp product (positions 360581). TG3 sense (5'-AGCCTGTGAACGTGCAGATGCTCTTC-3') and antisense (5'-TGATTGCAGGAAACTTGTTGCAGG-3') primers amplified a 225-bp product (positions 18672091). FXIIIA sense (5'-CCTGCGTACTCGAAGAGACC-3') and FXIIIA antisense (5'-CTTCGAACTGGCCATAGC tc-3') amplified a 188-bp product (positions 9931180).
TG Activity, PPi, Nucleotide Pyrophosphohydrolase (NPP), AP, and Cellular DNA AssaysCell-associated TG activity was determined by a modification of a previously described method (35). In brief, 0.02 mg of cell lysate was extracted and sonicated for 10 s in 5 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 0.2 mM MgSO4, 2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 0.4% Triton X-100, and was then loaded to Nunc-Immuno Module plates previously coated with 20 mg/ml N,N'-dimethylcasein. The lysates were incubated for 1 h in 100 mM Tris, pH 8.5, 20 mM CaCl2, 40 mM dithiothreitol, and 2 mM 5-(biotinamido)pentylamine (BP) (Pierce), and detection of the bound BP was performed as described (35).
Extracellular PPi was determined radiometrically and equalized for the DNA concentration in each well, as described (31). We determined specific activity of NPP and AP as described, with units of NPP and AP designated as moles of substrate hydrolyzed/h/g protein in each sample (31).
Statistical AnalysesWhere indicated, error bars represent S.D. Statistical analyses were performed using the Student's t test (paired 2-sample testing for means).
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RESULTS |
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Next, we assessed and compared TG catalytic activity in articular chondrocytes in response to ATRA, IL-1, and CNP. In unstimulated primary knee chondrocytes from the TG2 (+/+) and TG2 (/) mice we observed a
50% reduction in basal TG activity (Fig. 2A). IL-1
and ATRA treatment, but not CNP treatment, induced doubling of TG activity in primary TG2 (+/+) mouse articular chondrocytes (Fig. 2A). The respective findings for chondrocyte TG activity in response to the same agonists were confirmed when primary normal bovine knee articular chondrocytes were studied (Fig. 2B). In articular chondrocytes from TG2 (/) mice, IL-1
and ATRA failed to significantly increase TG activity (Fig. 2A). Thus, TG2 accounted for the majority of up-regulated TG2 catalytic activity in knee articular chondrocytes in response to both IL-1
and ATRA.
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Essential Role of TG2 in IL-1- and ATRA-induced Chondrocyte Matrix CalcificationWe confirmed that IL-1
and ATRA induced matrix calcification by chondrocytes, first using normal bovine knee cells carried for 14 days in a mineralization-promoting culture medium (Fig. 3A). Under the same conditions, CNP failed to significantly induce matrix calcification in bovine knee chondrocytes. To assess if the differential changes in matrix calcification were the result of altered generation of PPi, we measured extracellular PPi, which has been described to rise in association with chondrocyte hypertrophy (36). We also quantified cell-associated PPi-generating NPP activity (36), which modulates growth plate chondrocyte differentiation in vivo (37) (Fig. 3, BC). Under these conditions, CNP failed to significantly alter either PPi levels or NPP activity in bovine chondrocytes (Fig. 3, BC). We confirmed (38) that ATRA increased extracellular PPi and cell-associated NPP activity by 2-fold in normal bovine chondrocytes (Fig. 3, BC). We also confirmed (39) that IL-1
significantly decreased both extracellular PPi and NPP activity in the normal bovine chondrocytes. Thus, opposite changes in PPi generation occurred as a component of chondrocyte differentiation in response to ATRA and IL-1
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Parallel experiments using TG2 (+/+) articular chondrocytes treated with ATRA and IL-1 gave comparable results to bovine chondrocytes for stimulation of calcification, NPP-specific activity, and extracellular PPi (Fig. 4, AC). But in the absence of TG2, mouse chondrocytes stimulated with IL-1
or ATRA failed to carry out significantly increased matrix calcification relative to wild-type cells (Fig. 4A).
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ATRA increased extracellular PPi by more than 3-fold and NPP activity by more than 4-fold basal levels, and IL-1 significantly decreased both extracellular PPi and NPP activity in TG2 (+/+) mouse chondrocytes (Fig. 4, BC). TG2 (/) mouse chondrocytes had a less pronounced up-regulation of PPi and NPP in response to ATRA than in wild-type cells (Fig. 4, BC). But the TG2 (/) mouse chondrocytes significantly responded to ATRA and IL-1
with regulatory changes in extracellular PPi and NPP activity in the same respective directions as in wild-type cells (Fig. 4, BC). Because changes in PPi generation appeared less likely than TG2 to be a major force in driving calcification in response to ATRA and IL-1
, we next tested for potential effects of TG2 in modulating altered states of chondrocyte maturation differentially linked to calcification.
TG2-dependent and -independent Chondrocyte HypertrophyWe determined that ATRA-induced type X collagen expression, the cardinal marker for chondrocyte hypertrophy (40), was markedly reduced in primary TG2 (/) mouse chondrocytes (Fig. 5A). Induction by ATRA of additional markers of chondrocyte hypertrophy (i.e. AP activity (41) and MMP-13 expression (42)) also became attenuated in TG2 (-/-) chondrocytes (Figs. 5B and 6A). Yet TG2 null cells had the capacity to carry out induced expression and release of MMP-13, as there was no apparent difference in the induction of MMP-13 in response to IL-1 in TG2 (+/+) and TG2 (/) chondrocytes (Fig. 6B). Under these conditions, IL-1
did not induce type X collagen in TG2 (+/+) cells and only minimally induced type X collagen in TG2 (/) cells (Fig. 5A).
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CNP induced type X collagen expression (Fig. 7A), MMP-13 expression (Fig. 7B), and AP activity (Fig. 7C) in TG2 (+/+) chondrocytes. In contrast to the ATRA-treated cells, induction of each of these markers of chondrocyte hypertrophy by CNP was comparable in TG2 (/) and TG2 (+/+) chondrocytes. Thus, we tested if other ATRA-induced responses might be selectively impaired (relative to CNP responsiveness) in TG2 (/) mice. Specifically, we analyzed for potential impairments in TG2 (/) mouse chondrocytes of induction of the retinoic acid-responsive gene CD38 (43). We also evaluated induction of Cbfa1, whose functions in vivo include an essential role in promoting chondrocyte hypertrophy in vivo and hypertrophy and calcification in chondrocytes in vitro response to retinoic acid (44, 45). Concurrently, we tested for induction of the FXIIIA TG isozyme, because it is a marker of chondrocyte hypertrophy in vivo and directly stimulates chondrocyte calcification in vitro (19).
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ATRA induced CD38 mRNA expression in wild-type cells but not in TG2 (/) cells (Fig. 8). Both ATRA and CNP (but not IL-1) induced Cbfa1 mRNA expression in wild-type mouse chondrocytes. But in TG2 (/) chondrocytes, ATRA failed to induce Cbfa1 mRNA levels under conditions where the Cbfa1 mRNA up-regulation response to CNP was maintained (Fig. 8).
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We observed that ATRA, but not CNP (or IL-1), markedly induced FXIIIA in TG2 (+/+) mouse chondrocytes, as assessed by SDS-PAGE Western blot analysis of cell lysates (Fig. 9). In TG2 (/) mouse chondrocytes, the capacity for ATRA to induce FXIIIA was present, though substantially diminished (Fig. 9). In contrast, the capacity for CNP to induce FXIIIA was substantially up-regulated in TG2 (/) chondrocytes (Fig. 9). Hence, TG2 deficiency differentially altered chondrocyte hypertrophy and expression of certain mediators of calcification in response to ATRA and CNP. The results are summarized schematically in Fig. 10.
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DISCUSSION |
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TG catalytic activity was diminished by only 50% in unstimulated TG2 (/) mouse chondrocytes, and, in tandem with RT-PCR expression studies, our results suggested that both TG2 and FXIIIA normally contributed to basal TG transamidation catalytic activity in chondrocytes. In this context, FXIIIA is expressed as a latent enzyme. But chondrocyte-expressed proteolytic activity can stimulate FXIIIA activation, an event that may be accelerated in hypertrophic chondrocytes (49). The inflammatory cytokine IL-1
, a known contributor to the pathogenesis of OA (20), was confirmed (19) to induce both TG activity and matrix calcification in chondrocytes. But there was an absolute loss of a stimulated increase in either TG catalytic activity or calcification in response to IL-1
in the TG2 (/) mouse knee chondrocytes, under conditions where there was only a modest adaptive change in FXIIIA expression. Therefore, IL-1
-induced TG catalytic activity and calcification appeared to principally reflect essential, direct contributions of TG2.
Like IL-1, ATRA induced both TG catalytic activity and calcification. But ATRA, unlike IL-1
, markedly induced FXIIIA in wild-type mouse chondrocytes. Interestingly, ATRA remained able to induce FXIIIA in TG2 (/) cells, albeit to a lesser degree. Yet ATRA failed to induce either TG catalytic activity or calcification in TG2 (/) cells. Furthermore, ATRA-induced type collagen, MMP-13, and AP activity were attenuated in TG2 (/) cells, consistent with a block in development of chondrocyte hypertrophic differentiation. Moreover, CNP substantially induced FXIIIA in TG2 (/) cells in conjunction with CNP-induced hypertrophy but a lack of induction of calcification. Therefore, FXIIIA expression was subject to modulation via the presence or absence of TG2 in chondrocytes. It appeared unlikely that FXIIIA played a major role in either the development of articular chondrocyte hypertrophy in response to ATRA and CNP or in the induction of calcification by ATRA in vitro. But to establish or exclude specific contributions of FXIIIA to chondrocyte hypertrophy and calcification under stress, studies with genetically deficient FXIIIA chondrocytes will be necessary.
Cbfa1-mediated transcriptional activation of selected genes is a fundamental signal in the transition of chondrocytes from resting cells to terminally differentiated hypertrophic cells in physiologic endochondral development (44). Furthermore, ATRA is known to induce Cbfa1 expression, and Cbfa1 is essential for ATRA-induced chondrocyte maturation in vitro. We observed that ATRA failed to induce Cbfa1 in TG2 (/) chondrocytes, unlike the case for wild-type chondrocytes. Similarly, expression of CD38, a type II transmembrane glycoprotein with ecto-NAD+ glycohydrolase activity (43), was induced in TG2 (+/+) but not in TG2 (/) chondrocytes. Yet basal levels of both PPi and NPP activity were unaltered in the TG2 (/) cells. In addition, ATRA remained able to significantly induce PPi and NPP levels (as well as FXIIIA expression cited above) in the absence of TG2, albeit to a lesser extent than in TGs (+/+) chondrocytes. Taken together, our data suggested that TG2 exerted differential regulatory effects on ATRA-induced signaling in chondrocytes.
The actions in higher eukaryotes of ATRA and other vitamin A-derived metabolites are mediated via direct binding to RARs and RXRs, which are members of the nuclear hormone superfamily. The transcriptional activating effects of these retinoid receptors are regulated by conformational changes triggered by hormone binding to structurally conserved ligand-binding domains of these receptors (50). In this study, we limited our investigation of retinoids to effects of the RAR agonist ATRA and included assessment of expression of CD38 expression, which is well characterized to be RAR-mediated (43). But it should be noted that differential expression of distinct retinoid receptor subtypes and their isoforms, as well as homodimerization and heterodimerization of retinoid receptors with each other and other nuclear hormone receptors, confer signaling specificity between retinoids and divergent signaling mediated by individual retinoids (51). Such effects could have contributed to the differential effects of TG2 on ATRA-induced gene expression and functional responses in chondrocytes in this study. RAR and RXR ligands both can induce TG activity and can stimulate MMP-13 expression in primary chondrocytes (52). Therefore, it will be of interest to further dissect the effects of TG2 on retinoid signaling in chondrocytes using agonists and antagonists of specific RARS and RXRs.
In this study, ATRA and IL-1 both induced transamidation activity in a TG2-dependent manner in chondrocytes. However, further study will be required to define the subcellular location(s) where TG2 acts to regulate retinoid signaling, gene expression, differentiation, and calcification, as well as the role of TG2 transamidation activity in each of these TG2 functions in chondrocytes. TG2 is constitutively localized in the cytosol (16), and, under certain conditions, effects of TG2 on intracellular TG substrates can restrain apoptosis (52) and thereby potentially allow for progressive alterations in differentiation in stressed cells. Though TG2 lacks a signal peptide, it can be externalized, which allows cell surface TG2 to potentially mediate cell binding to fibronectin in a direct manner via the TG2 N-terminal fibronectin binding domain (22). Through cell adhesion-modulating activities, including potential TG2-induced cross-linking of substrate matrix proteins, TG2 might influence signal transduction and differentiation in chondrocyte by modulating how the matrix communicates with the cell (15). By catalyzing formation of intramolecular cross-links, TG2 can also enhance the activity of phospholipase A2 and thereby mediate inflammation (53). Alternatively, the capacity of TG2 to regulate Phospholipase C d1 signaling (22), and signal transduction and Pi generation through GTPase and ATPase activities of TG2 (22), could modulate TG2 involvement in retinoid signaling, hypertrophy, and calcification in chondrocytes. In this regard, it is noteworthy that the accumulation of intracellular Pi at critical times during chondrocyte differentiation induces mineralization and increases expression of Cbfa1 and type X collagen (54).
In this study, we demonstrated that CNP was able to induce Cbfa1 expression and hypertrophy (evidenced by up-regulated type X collagen and MMP-13 expression as well as AP activity) but not calcification in cultured articular chondrocytes. In mice with targeted disruption of CNP, dwarfism of the achondroplastic type occurs in which there is decreased height of the growth plate, particularly in the hypertrophic zone (25). Because the CNP-deficient phenotype cannot be rescued by cyclic GMP-dependent protein kinase II, ligand-specific stimulation by CNP of GC-B-induced cGMP generation and downstream cyclic GMP-dependent protein kinase II activation appear to be centrally involved in the effects of CNP on chondrocyte development and maturation (55). In this study, CNP-induced Cbfa1 expression and articular chondrocyte hypertrophy did not require TG2, consistent with internal redundancy in mechanisms promoting chondrocyte hypertrophy. But it remains to be determined if up-regulated TG2 expression by chondrocytes could partially overcome defects in chondrocyte maturation due to CNP deficiency.
In conclusion, we have demonstrated that TG2 was an essential mediator of chondrocyte hypertrophy and calcification in response to ATRA, as well as calcification that occurred without hypertrophy in response to IL-1. CNP also induced hypertrophy in a TG2-independent manner without calcification in articular chondrocytes. These findings indicate means by which hypertrophy and calcification may be dissociated in chondrocytes. Furthermore, the existence of redundant TG2-dependent and TG2-independent mechanisms for chondrocytic cells to develop hypertrophy likely explains why normal TG2 null mice demonstrate no gross phenotypic abnormalities in their growth plates or skeletons. Tissue forms of TGs are held to play major roles as repair enzymes for tissue injury, in part by modulating matrix stability through protein cross-linking (48, 56). The results of this study suggest a pathophysiologic role of up-regulated TG2 activity in promoting premature epiphyseal closure and pathologic calcification associated with hypervitaminosis A and certain forms of systemic retinoid toxicity (28). In addition, TG2 may be a significant factor in promoting calcification in injured chondrocytes in OA cartilage, which can contain known inducers of chondrocyte TG activity such as IL-1
, tumor necrosis factor-
, nitric oxide, and peroxynitrite (19). TG2 activity also may mediate development of chondrocyte hypertrophy and calcification in response to the chemokines IL-8 and Growth-related oncogene
(GRO
), which can be expressed in OA cartilage (57). Therefore, our results suggest that TG2 may provide a novel site for therapeutic intervention in certain forms of pathologic cartilage calcification.
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FOOTNOTES |
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¶ To whom correspondence and reprint requests should be addressed: VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161. Tel.: 858-552-8585 (ext. 3519); Fax: 858-552-7425; E-mail: rterkeltaub{at}ucsd.edu.
1 The abbreviations used are: OA, osteoarthritis; AP, alkaline phosphatase; ATRA, all-trans form of retinoic acid; Cbfa1, core binding factor alpha 1; CNP, C-type natriuretic peptide; NPP, nucleotide pyrophosphatase phosphodiesterase; RAR, retinoic acid receptor; RXR, retinoid X receptor; TG, transglutaminase; RT, reverse transcription; FXIIIA, factor XIIIA.
2 R. Graham, N. Nanda, and J. Eisman, unpublished observations.
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REFERENCES |
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