- Email: [email protected]
TISSUE TRANSGLUTAMINASE EXPRESSION IN QUAIL EPIPHYSEAL CHONDROCYTES
Cell Biology International 1998, Vol. 23, No. 1, 41–49 Article No. cbir.1998.0316, available online at http://www.idealibrary.com on
TISSUE TRANSGLUTAMINASE EXPRESSION IN QUAIL EPIPHYSEAL CHONDROCYTES ELISA GIONTI1,3*, MASSIMO SANCHEZ2, ANTONIETTA ARCELLA3, GIANFRANCO PONTARELLI3, SIMONA TAVASSI4, VITTORIO GENTILE4, ANNA COZZOLINO4 and RAFFAELE PORTA5 Dipartimento di Medicina Clinica e Sperimentale, Via T. Campanella, 88100 Catanzaro, Italy; 2Istituto Superiore di Sanita`, viale Regina Elena, 299–00161 Roma, Italy; 3Dipartimento di Biochimica e Biotecnologie Mediche, Universita` di Napoli ‘Federico II’, Via S. Pansini, 5–80131 Napoli, Italy; 4Dipartimento di Biochimica e Biofisica, II Universita` di Napoli, Via Costantinopoli, 16–80138 Napoli, Italy; 5CIRPEB Centro Interdipartimentale di Ricerca sui peptidi bioattivi, Universita` di Napoli ‘‘Federico II’’, via Mezzocannone, 4, 80138 Napoli, Italy
1
Received 31 March 1998; accepted 25 September 1998
Tissue transglutaminase (tTGase) is a GTP-binding Ca2+ -dependent enzyme which catalyses the post-translational modification via å(ã-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme. tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation. tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation. Our findings lend support to the idea that tTGase might play a role in non-dividing cultured 1999 Academic Press chondrocytes. K: retinoic acid; tissue transglutaminase; cell adhesion; cell growth; chondrocyte phenotype.
INTRODUCTION Transglutaminases (TGases, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyse post-translational modifications of proteins by the introduction of an isopeptide bond between the ã-carboxamide group of proteinbound glutamines and the å-amino group of protein-bound lysines (for reviews see Folk, 1980; Lorand and Conrad, 1984; Greenberg et al., 1991; Aeschlimann and Paulsson, 1994). Various transglutaminases have been characterized both in *To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Via S. Pansini, 5-80131 Naples, Italy; E-mail: [email protected] 1065–6995/99/010041+09 $30.00/0
intracellular and extracellular compartments (Aeschlimann and Paulsson, 1994). Tissue transglutaminase (tTGase) is a predominantly cytosolic enzyme whose expression is regulated by all transretinoic acid (RA) in vivo and in vitro. Induction of tTGase occurs soon after the addition of RA; the mediators of RA-induced tTGase expression are the nuclear receptors for RA, RAR-á, RAR-â and RAR-ã, as well as the nuclear receptors for 9-cisretinoic acid, RXR’s (Moore et al., 1984; Chiocca et al., 1988; Nara et al., 1989; Piacentini et al., 1992; Nakanishi et al., 1991; Zhang et al., 1995; Nagy et al., 1995). The physiological function of tissue transglutaminase has been associated with the activation of the programmed cell death 1999 Academic Press
42
process (apoptosis) (Fesus et al., 1991; Nagy et al., 1995). The enzyme, however, can also be associated with the cell membrane to exert a receptor-coupled signaling function (Nakaoka et al., 1994; Mian et al., 1995; Chen et al., 1996) or expressed at the extracellular surface of many cells (Barsigian et al., 1991; Martinez et al., 1994). Several extracellular matrix components are tTGase substrates (for review see: Aeschlimann and Paulsson, 1994). In fibroblasts and in endothelial cells, the enzyme plays a role in the extracellular matrix stabilization (Martinez et al., 1994), cell adhesion and cell morphology (Gentile et al., 1992; Jones et al., 1997). The expression of tTGase in cartilage tissues correlates with the terminal differentiation of chondrocytes (Aeschlimann et al., 1993). Cartilage development proceeds through distinct stages starting from mesenchymal cell condensation and resulting in the differentiation of resting chondrocytes. During endochondral ossification, resting chondrocytes undergo a further process of differentiation/maturation involving proliferation, hypertrophy, calcification, degradation and replacement of cartilage by bone and marrow (for reviews see Poole, 1991; Sandberg, 1991; Hunziker, 1994; Erlebacher et al., 1995). In developing long bones tTGase expression correlates with chondrocyte maturation (Aeschlimann et al., 1993). The enzyme is externalized from hypertrophic chondrocytes and might be involved in matrix cross-linking before cartilage undergoes calcification. Furthermore, osteonectin and collagen II are target proteins in hypertrophic chondrocytes (Aeschlimann et al., 1993; Hohenaldl et al., 1995). Extracellular matrix of cartilage contains a variety of proteins including cartilage specific proteoglycan aggrecan, collagen II, tenascin and several minor collagens (Hascall, 1988; van der Rest and Garrone, 1991; Mayne and Brewton, 1993; Mackie et al., 1987). The expression of genes for extracellular matrix components varies during cartilage differentiation/maturation process (for reviews see Poole, 1991; Sandberg, 1991; Hunziker, 1994; Erlebacher et al., 1995). Mesenchymal cells produce collagens I and III; chondrocytes synthesize aggrecan and collagens II, IX, XI and X. The phenotype of cartilage cells undergoes extensive modulation in cell culture as well and the mechanisms regulating the differential expression of extracellular matrix genes are largely unknown (for reviews see Ramirez and Di Liberto, 1990; Petit et al., 1992). Retinoids are a well established means of studying the regulation of extracellular matrix genes in cultured chondrocytes (Benya and Padilla,
Cell Biology International, Vol. 23, No. 1, 1999
1986; Horton et al., 1987; Oettinger and Pacifici, 1990). Primary cultures of quail epiphyseal chondrocytes synthesize the cartilage-specific collagens type II and type IX. Treatment of suspension chondrocytes with retinoic acid inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression (Sanchez et al., 1991, 1993). RA-induced inhibition of the chondrocyte phenotype correlates with cell adhesion. In fact, RA-chondrocytes undergo their phenotypic modulation as they begin to attach. Further, prevention of cell adhesion blocks many of the effects on the chondrocyte phenotype induced by RA (Sanchez et al., 1996). Here we used primary cultures and a permanent cell line of chondrocytes to study tissue transglutaminase expression and its relationships with cell proliferation, cell differentiation and cell adhesion.
MATERIALS AND METHODS Cell culture Primary chondrocytes were isolated from day 10 quail embryo tibiae as previously described (Gionti et al., 1985) with the following modifications. Tibiae were incubated for 1 h in a saline solution containing trypsin/collagenase and the cells released after this incubation are discarded as they consisted mainly of perichondrial cells. Epiphysis were dissected, minced and incubated at 37C in fresh enzyme mixture; cells released by sequential enzymatic digestion from about 50 embryos were pooled and seeded at low initial cell density (2000 cells/cm2). Floating cells were resuspended in Coon’s modified F12 medium (AmbesiImpiombato et al., 1980) supplemented with 10% foetal calf serum. Expression of the differentiated phenotype was assessed by SDS-PAGE of metabolically labeled proteins. all trans-RA (generously provided by Hoffmann-La Roche) was dissolved in 95% ethanol and stored at 80C in the dark. On day 0, this solution was diluted with growth medium and control cultures received an equivalent amount of ethanol. After addition of 0.5 ì RA, cells were plated at 1105 cells/ml. No toxic effect was ever detected with this concentration of RA in treated cultures. All the experiments described in this report were carried out with ordinary foetal calf serum. Parallel experiments in which delipidised serum was used in either control or RA treated cultures did not show significant differences
Cell Biology International, Vol. 23, No. 1, 1999
in tissue transglutaminase activities. To prevent RA-induced cell adhesion, culture dishes were coated with a film of agarose. The continuous cell line MC2 was derived from quail epiphyseal chondrocytes infected with avian myelocitomatosis virus/Rous associated virus-1, subgroup A -MC29(RAV-1)-, a retrovirus carrying the v-myc oncogene (Gionti et al., 1985). Primary epiphyseal chondrocytes can grow either in suspension or in monolayer. Because of their higher sensitivity to viral infection, chondrocytes grown in monolayer were originally used in infection experiments; here, monolayer cells are used as the normal counterpart of the infected cell line. DNA probes The tTGase probe was a full-length clone harbouring a 3200-bp cDNA insert isolated from a cDNA library made from chicken heart (kindly provided by Drs P. Davies and V. Thomazy, Houston, TX, U.S.A.). The coding and the 3 untranslated region shows high homology to the cDNA clone isolated by Weraarchakul-Boonmark et al. (1992). The RAR-â2 probe used was g RAR-â, harbouring a 1600-bp cDNA insert including the entire coding region and the 3 untranslated region of the chicken gene (Smith and Eichele, 1991). The GAPDH probe was a full-length cDNA clone (1200 bp) encoding rat glyceraldehyde-3-phosphate dehydrogenase (Fort et al., 1985). The inserts of these plasmids were used as probes. High specific activity random primed probes were prepared with the Promega kit as specified by the supplier. RNA extraction and Northern blot hybridization Total cellular RNA (2107 cells/sample) extracted as previously described (Chomczynski and Sacchi, 1987), was denatured, fractionated on formaldehyde/agarose gels (Lehrach et al., 1977) and blotted onto an Amersham Hybond N nylon membrane. Pre-hybridization and hybridization were performed in 500 m NaH2PO4 pH 7.2, 7% SDS, 1 m EDTA for 30 min and 16 h, respectively; the hybridization temperature was 65C. Filters were washed three times at 65C in 50 m NaH2PO4 pH 7.2, 1% SDS. Washed filters were dried and exposed to Fuji films with intensifying screens at 80C. Densitometric scanning analysis was performed using software from NIH-Image. Transglutaminase assay Cells (3106 cells/sample) were mechanically removed from dishes, rinsed twice with PBS, resus-
43
pended and homogenized in Tris–HCl 20 m (pH 7.4) containing 2 m EGTA, pepstatin (0.1 ìg/ 100 ìl), leupeptin (0.1 ìg/100 ìl), PMSF 0.1 m, bestatin (0.1 ìg/100 ìl) and sonicated at 4C for 20 s. Transglutaminase activity was measured by detecting the incorporation of [3H]spermidine into N,N -dimethylcasein as previously reported (Lorand, 1972). The incubation mixture contained 125 m Tris–HCl (pH 8.0), 2.5 m CaCl2, 10 m dithiothreitol (DTT), 200 ìg N,N -dimethylcasein, 12.5 ì spermidine containing 0.15 ìCi [3H]spermidine and 5 to 25 ìg of protein from total cell homogenate in a final volume of 0.1 ml. After 30 min incubation at 37C, the samples were precipitated with 10% TCA. Free [3H]spermidine was eliminated by washing with large volumes of cold 10% TCA containing 1 m spermidine. The pellet was resuspended and bound [3H]spermidine was counted with 5 ml of Pico Fluor 30 scintillation cocktail (United Technologies/Packard). Chondrocytes enzyme activity was inhibited by adding EGTA thus suggesting that it is calciumdependent. Western blot analysis Determination of tissue transglutaminase protein was carried out with condrocytes lysates by Western blot analysis. tTGase-positive bands were revealed by using as a primary antibody an affinity purified IgG raised in goat against purified guinea pig liver tTGase proven to be not cross-reactive with other forms of transglutaminase (Chiocca et al., 1988). Statistical analysis Statistical significance of tTGase mRNA levels between control and RA-treated chondrocytes were calculated using Student’s t-test. RESULTS Retinoic acid stimulates tTGase expression in primary chondrocytes Like chondrocytes from many species, quail epiphyseal chondrocytes (QEC) can grow either in monolayer as polygonal cells or in suspension. Since suspension quail chondrocytes convert poorly into attached cells, they are a suitable system in which to study the alterations in cellmatrix interactions that occur during RA-induced changes in cell shape (Sanchez et al., 1991).
44
Cell Biology International, Vol. 23, No. 1, 1999
Fig. 1. Morphology of control and RA treated primary chondrocytes. A: control untreated chondrocytes (the picture was taken 3 days after plating); B, C and D: chondrocytes treated with 0.5 ì RA for 1, 2 and 3 days, respectively. Cells were photographed under phase contrast: magnification 75.
Exposure of suspension QEC to RA for three days blocks cell growth, inhibits the expression of cartilage-specific collagens type II and IX, turns on fibronectin expression and induces cell adhesion (Sanchez et al., 1991, 1996). One day after RA addition about 50% of the cells adhere to the substrate; by 2 days of treatment adhesion is almost complete (Fig. 1). By 3 days many cells display a dedifferentiated flattened morphology consistent with the results obtained by Hassel et al. (1979). Transglutaminase activity was found in chondrocyte lysates (Table 1). Enzyme activity increases upon RA treatment and reaches its maximum after around 16 h of treatment then gradually declines to slightly lower values than the base-line activity detected in control chondrocytes. Stimulation of enzyme activity is accompanied by parallel and equivalent increases of a 77-kDa band as detected by Western blot analysis in cell lysates from control and RA treated chondrocytes (not shown). A single 3.2-kb band was detected by hybridization of total RNA from suspension QEC with a cDNA clone encoding chicken tissue-transglutaminase (Fig. 2). In the experiment shown in Figure 2, stimulation of tTGase expression occurs between 4 and 8 h after RA addition and remains unchanged over the subsequent 16 h. Thereafter, tTGase mRNA levels decline until at 72 h they reach the basal levels detected in untreated control chondrocytes (the results are not shown but have been made available
to the referees). RAR-â2 expression is readily induced upon RA addition (Fig. 2), consistently with the results obtained in chicken sternal chondrocytes by Iwamoto et al. (1993a). RA-treated chondrocytes display a 2–4-fold increase of tTGase mRNA over the levels detected in untreated control chondrocytes as evaluated in different experiments by densitometric scanning analysis and normalization to GAPDH expression (Table 2). Cell adhesion does not correlate with tTGase expression in untreated chondrocytes: similar levels of tTGase expression were observed in Table 1. Transglutaminase activity in chondrocyte homogenates from primary cultures treated with 0.5 ì RA for the indicated times Treatment (h)
Control 8 16 24 40 72
Transglutaminase activity* (pmoles of [3H]spermidine incorporated/mg protein) 26.00.49 45.50.88 66.91.37 52.31.21 43.80.71 15.61.4
*Values represent the mean value of three different experiments carried out by triplicate determinations.. Further experimental details are reported in the Materials and Methods.
Cell Biology International, Vol. 23, No. 1, 1999
45
Fig. 2. Time course of RA-induced stimulation of tissue transglutaminase and RAR-â2 genes in primary cultures of quail epiphyseal chondrocytes. Steady-state levels of tTGase, RAR-â2 and GAPDH mRNAs in control chondrocytes (lane 1) and in chondrocytes treated with 0.5 ì RA for 4, 8 and 24 h (lanes 2, 3 and 4, respectively).
untreated cultures growing either in suspension or in monolayer as judged by Northern blot (Fig. 2, lane 1; Fig. 3, lane 1), by Western blot analysis and by enzyme activity assays (not shown). To investigate whether the stimulation of tTGase expression correlates with RA-induced cell Table 2. Relative levels of tTGase mRNA (fold of stimulation) in RA-treated chondrocytes
Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7 Means ..
Monolayer
Suspension
3.5 1.6 3.45 4.4 — — 3 3.19** 1.023719
2.75 — — 2.25 4.8 2.75 — 3.1375* 1.133119
Primary chondrocytes were cultured either in permissive conditions for RA-induced cell adhesion (Monolayer) or in non-permissive conditions for RA-induced cell adhesion (Suspension). Total RNAs extracted from control and RA-treated chondrocytes were isolated and analysed by Northern blot for tTGase expression. tTGase and GAPDH bands were scanned and the scan units were converted into -fold stimulation over the basal levels detected in control chondrocytes. In the experiments 1, 3, 6, the reported values refer to stimulations obtained after 6 h of RA-treatment; in the other experiments (2, 4, 5, 7) the reported values refer to shorter treatments (4 h). Significant differences from controls are designated as *P<0.05, **P<0.01. —, Not done.
Fig. 3. Tissue transglutaminase responsiveness to RA treatment in primary chondrocytes and MC2, a chondrocyte cell line immortalized by the activated form of Myc. Steady-state mRNA levels of tTGase and GAPDH mRNAs in untreated primary chondrocytes grown in monolayer (lane 1); primary chondrocytes treated with RA for 8 h (lane 2); in untreated MC2 cells (lane 3) and MC2 cells treated with RA for 8 h and 24 h (lanes 4 and 5, respectively). Because of their higher sensitivity to viral infection, primary chondrocytes grown in monolayer have been originally used in the infection experiments; here they are used as the normal counterpart of the infected cell line.
adhesion, we prevented cell adhesion by culturing chondrocytes on agarose-coated dishes. We have previously shown that RA treatment does not significantly affect the expression of the chondrocyte phenotype if cell adhesion is prevented (Sanchez et al., 1996). Stimulation of tTGase expression occurs in non permissive conditions for RA-induced cell adhesion (Table 2), while the RA treatment fails to inhibit the chondrocyte phenotype as assessed by SDS-PAGE of metabolically labeled proteins (not shown). Equivalent increases of enzyme activity and tTGase protein levels also occur (not shown). RA treatment blocks cell proliferation both in permissive and in non permissive conditions for cell adhesion (Sanchez et al., 1996). Cell survival was assayed at the end of RA treatment: most of the cells (90%) retained the ability to exclude Trypan Blue, regardlesss of culture conditions. tTGase expression in MC2, a cell line derived from retrovirus infected chondrocytes We next analysed the tTGase expression in a chondrocyte cell line we have established in our laboratory from primary QEC infected with avian myelocytomatosis virus -MC29 (RAV-1)-, a v-myccarrying retrovirus (Gionti et al. 1985). Infection of monolayer QEC with MC29 (RAV-1) does not induce cell transformation, stimulates cell proliferation and leads to the establishment of a continuous cell line, designated as MC2, which maintains the expression of cartilage differentiation markers besides the typical polygonal morphology of chondrocytes growing in monolayer (Gionti et al., 1985).
46
Differently from uninfected chondrocytes, we could not observe tTGase transcripts in MC2 cells and RA treatment fails to activate tTGase expression in these cells as assessed by Northern blot (Fig. 3). Prolonged exposure to RA has no significant effect on tTGase expression (not shown). The rate of proliferation of MC2 cells is 50% inhibited after a 3d treatment. RAR-â2 expression is readily induced by RA treatment in MC2 cells as assessed by Northern blot analysis (not shown). DISCUSSION Here we report that tTGase activity is present in primary cultures of quail epiphyseal chondrocytes (QEC) and increases transiently after RA treatment. Increase in steady-state levels of tTGase mRNA occurs between 4 and 8 h of RA treatment. Conversely, the inhibition of cartilage phenotypic markers and changes in cell morphology we observe in RA treated cultures become apparent only after several hours or even days of exposure to RA (Sanchez et al. 1991, 1993, 1996). Such delayed responses likely result from changes in gene expression secondary to the direct action of RA and its receptors. In vivo, tissue transglutaminase distribution correlates with chondrocyte maturation (Aeschlimann et al. 1993). Chondrocyte maturation is the complex developmental process starting after chondrocyte differentiation and resulting in mature hypertrophic chondrocytes which produce an extracellular matrix permissive for calcification (for reviews see: Poole, 1991; Hunziker, 1994). In permanent cartilage, e.g. normal articular cartilage, chondrocyte maturation does not take place but it is interrupted at the stage of resting chondrocytes. tTGase activity is accumulated intracellularly in the proliferation/maturation zone and it is externalized by hypertrophic chondrocytes before cartilage undergoes calcification (Aeschlimann et al., 1993). The transglutaminase reaction products, the ã-glutamyl-å-lysine cross-links, are abundant in the mineralizing cartilage matrix during the endochondral bone formation and the maturation of cartilages that do not undergo ossification, e.g., tracheal and laryngeal cartilages (Aeschlimann et al., 1995). Osteonectin and collagen II are target proteins for cross-linking in hypertrophic chondrocytes (Aeschlimann et al., 1995; Hohenaldl et al., 1995). The enzyme responsible for cross-linking of the mineralizing cartilage matrix is tTGase while it remains unclear which transglutaminase is expressed in osteoblasts (Aeschlimann et al. 1996).
Cell Biology International, Vol. 23, No. 1, 1999
Numirskaya and Linsenmayer (1996) identified in cultured hypertrophic chondrocytes a chicken homologue of the human Factor XIIIa which was upregulated 3–4-fold over non-hypertrophic chondrocytes. These findings are not consistent with the immunohistochemical identification of the cartilage molecule as tTGase (Aeschlimann et al. 1993). Perhaps this discrepancy was due to aspecific activation of gene expression produced by the in vitro environment. Alternatively, hypertrophic chondrocytes may express more than one form of transglutaminase. Two forms of transglutaminase activity that differed in their cellular localization were reported in cultured articular chondrocytes from rabbits (Demignot et al., 1995) and from adult pigs (Rosenthal et al., 1997) while Aeschlimann et al. (1993) found no TGase activity in articular cartilages from rats and calves. Rabbit articular chondrocytes readily dedifferentiate upon subculture (Benya and Padilla, 1986); similarly, transglutaminase activity drops at the second passage. TGase activity in rabbit articular chondrocytes was mainly membrane associated and was downregulated by RA treatment (Demignot et al., 1995) while the cytosolic activity was identified as tTGase (Borge et al., 1996). The present report dealing with tTGase activity in primary QEC, which display a differentiated phenotype upon subculture, provides further support to the idea that tTGase activity has a physiological role in cartilage cells. Upregulation of tTGase by RA is well established (Moore et al., 1984; Chiocca et al., 1988; Nara et al., 1989; Piacentini et al., 1992; Gentile et al., 1992) and RA has been shown to play an important role in the regulation of chondrocyte maturation and cartilage mineralization (Iwamoto et al., 1993b; Ballock et al., 1994). RA-induced inhibition of the differentiation programme in suspension QEC is associated with growth arrest and induction of cell adhesion (Sanchez et al., 1991, 1996). Increased enzyme activity might lead to cross-linking of extracellular matrix proteins thus contributing to matrix remodeling that occurs upon RA-induced cell adhesion. It has been suggested that in other cell types increased tTGase activity correlates with morphological changes (Byrd and Licthi, 1987; Nara et al., 1989: Nakanishi et al., 1991) and increased cell adhesion (Cai et al., 1991; Gentile et al., 1992; Jones et al., 1997). In quail epiphyseal chondrocytes, RAinduced cell adhesion correlates with the inhibition of the expression of chondrocyte phenotypic traits. In fact, we have observed that prevention of cell
Cell Biology International, Vol. 23, No. 1, 1999
adhesion results in the failure of the RA-induced inhibition of the chondrocyte phenotype (Sanchez et al., 1996). In culture conditions non-permissive for cell adhesion we still observe RA-induced stimulation of tTGase expression. Therefore, stimulation of tTGase by RA can be uncoupled from the inhibition of the chondrocyte phenotype by RA which, in contrast, is dependent on cell adhesion and cell shape changes. These results suggest that stimulation of tTGase expression in QEC might result from a more direct effect of RA and its receptors on the regulation of this gene rather than being subsequent to cell adhesion. Borge et al. (1996) showed that tTGase expression in rabbit articular chondrocytes correlates with cell adhesion and spreading regardless of their differentiation state; conversely, in quail epihyseal chondrocytes tTGase gene is expressed in both suspension and monolayer cultures and stimulation by RA occurs independently of cell adhesion. In considering these discrepancies, it must be noted that there are several important variations in the study we now report. In addition to the differences in the species and origin of the cells, dedifferentiation of rabbit articular chondrocytes starts as soon as the first passage (Demignot et al., 1995), while QEC display a differentiated phenotype upon prolonged culture (Sanchez et al., 1996). Whether these discrepancies relate to the maturation state of the responding cells in these two culture systems or they pertain to some other phenomena, remains to be determined. Our data show that RA-induced stimulation of tTGase expression in primary cultures of chondrocytes correlates with cell growth suppression. It has been reported that inhibition of transglutaminase activity results in the stimulation of cell proliferation in WI-38, a lung cell line (Birckbichler et al., 1981). Further, reduced levels of transglutaminase have been observed in tumors (Barnes et al., 1985; Knight et al., 1991) and in transformed cells (Birckbichler et al., 1977; Kosa et al., 1993). Here we report the loss of tTGase expression in a continuously growing chondrocyte cell line and an increased gene expression in growth arrested primary chondrocytes. It has been proposed that tTGase activity might play a role in the control of cell growth (Birckbichler et al., 1981; Mian et al., 1995). In this regard, our observations support this hypothesis. Lu and Davies (1997) have shown that alterations in DNA methylation of tTGase promoter may be one of the mechanisms regulating the expression of this gene. Whether the loss of tTGase expression in the chondrocyte cell line correlates with hypermethylation of the tTGase promoter
47
or with some other mechanisms remains to be elucidated. An understanding of the biological role of tTGase in cartilage cells will further our understanding of the complex molecular mechanisms underlying the control of cell growth and differentiation in this cell lineage.
ACKNOWLEDGEMENTS We are much indebted with Dr P. J. A. Davies and Dr V. Thomazy (Dept. of Pharmacology, Medical School, University of Texas, Houston) for providing us with the cDNA clone for chicken tTGase used in this study. Dr P. J. A. Davies has also provided the tTGase antibody. The financial support from MURST 40% ‘Meccanismi di Cancerogenesi’, PRIN ‘Struttura e regolazione dell’espressione dei geni in eucarioti’ and TelethonItaly grant #E738 is gratefully acknowledged. We thank V. Brescia for technical assistance. REFERENCES A D, W A, F H, P M, 1993. Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation in condrocytes. J Cell Biol 120: 1461–1470. A D, P M, 1994. Transglutaminases: protein cross-linking enzymes in tissue and body fluids. Thromb Haemost 71: 402–415. A D, K O, P M, 1995. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J Cell Biol 129: 881–892. A D, M D, P M, 1996. Tissuetransglutaminase and Factor XIII in cartilage and bone remodeling. Semin Thromb Hemost 22: 437–443. A-I FS, P LAM, C HG, 1980. Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA 77: 3455–3459. B RT, H A, W LM, F KC, R AB, S MB, 1994. Inhibition of chondrocyte phenotype by retinoic acid involves up-regulation of metalloprotease genes independent of TGF-â. J Cell Physiol 159: 340–346. B RN, B PJ, E BM, W PL, G M, 1985. Alterations in the distribution and activity of transglutaminase during tumor growth and metastasis. Carcinogenesis 6: 459–463. B C, S AM, M J, 1991. Tissue transglutaminase covalently incorporates itself, fibrinogen or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. J Biol Chem 266: 22,501–22,509. B PD, P SR, 1986. Modulation of rabbit chondrocyte phenotype by retinoic acid terminates type II collagen
48
synthesis without inducing type I collagen: the modulated phenotype differs from that produced by subculture. Dev Biol 112: 296–305. B PJ, O GR, C E, P MK, 1977. Transglutaminase activity in normal and transformed cells. Cancer Res 37: 1340–1344. B PJ, O GR, P MK, C E, C HA, 1981. Increase in proliferative markers after inhibition of transglutaminase. Proc Natl Acad Sci USA 78: 5005–5008. B L, D S, A M, 1996. Type II transglutaminase expression in rabbit articular chondrocytes in culture: relation with cell differentiation, cell growth, cell adhesion and cell apoptosis. Biochim Biophys Acta 1312: 117–124. B JC, L U, 1987. Two types of transglutaminase in PC12 pheochromocytoma cell line. J Biol Chem 262: 11,699–11,705. C D, B T, D L LM, 1991. Retinoids induce transglutaminase in NIH 3T3 cells. Biochem Biophys Res Commun 175: 1119–1124. C S, L F, I S, L KN, B PJ, G RM, 1996. á1-adrenergic receptor signaling via Gh is subtype specific and independent of its transglutaminase activity. J Biol Chem 271: 32,385–32,391. C EA, D PJA, S JP, 1988. The molecular basis of retinoic acid action: transcriptional regulation of tissue transglutaminase gene expression in macrophage. J Biol Chem 263: 11,584–11,589. C P, S N, 1987. Single step method of RNA isolation by acid guanidium thiocianate-phenol-chloroform extraction. Anal Biochem 162: 156–159. D S, B L, A M, 1995. Transglutaminase activity in rabbit articular chondrocytes in culture. Biochim Biophys Acta 1266: 163–170. E A, F EH, G SE, D R, 1995. Toward a molecular understanding of skeletal development. Cell 80: 371–378. F L, D PJA, P M, 1991. Apoptosis: molecular mechanisms in programmed cell death. Eur J Cell Biol 56: 170–177. F JE, 1980. Transglutaminases. Annu Rev Biochem 49: 517–531. F P, M L, P M, S SE, D C, J P, B JM, 1985. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigenic family. Nucl Acid Res 13: 1431–1442. G V, T V, P M, F L, D PJA, 1992. Expression of tissue transglutaminase in Balb-c 3T3 fibroblasts: Effects on cellular morphology and adhesion. J Cell Biol 119: 463–474. G E, P G, C R, 1985. Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes. Proc Natl Acad Sci USA 82: 2756–2760. G CS, B PJ, R RH, 1991. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J 5: 3071–3077. H VC, 1988. Proteoglycans: the chondroitin sulfate/ keratan sulfate proteoglycan of cartilage. ISI Atlas Sci (Biochem): 189–198. H JR, P JP, K HK, P RM, Y KM, 1979. Enhanced cellular fibronectin accumulation in chondrocytes treated with vitamin A. Cell 17: 821–826.
Cell Biology International, Vol. 23, No. 1, 1999
H C, M K, M U, T R, P M, A D, 1995. Two adjacent N-terminal glutamines of BM-40 (Osteonectin-SPARC) act as amine acceptor sites in transglutaminasec-catalyzed modification. J Biol Chem 270: 23,415–23,420. H WE, Y Y, H JR, 1987. Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes. Dev Biol 123: 508–516. H EB, 1994. Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Technique 28: 505–519. I M, G EB, A SL, N S, P M, 1993a. Responsiveness to retinoic acid changes during chondrocyte maturation. Exp Cell Res 205: 213–224. I M, S IM, Y K, B AL, L PS, A, SL, P M, 1993b. Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes. Exp Cell Res 207: 413–420. J RA, N B, M S, D PJA, G M, 1997. Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin. J Cell Sci 110: 2461–2472. K CRL, R RC, G M, 1991. Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumor progression. Biochem Biophys Acta 1096: 312–318. K K, M K, D L LM, 1993. Transformation of NIH3T3 cells with ras oncogenes abrogates the retinoic acid induction of tissue transglutaminase. Biochem Biophys Res Commun 196: 1025–1033. L H, D D, W J, B H, 1977. RNA molecular weight determination by gel electrophoresis under denaturing conditions. A critical reexamination. Biochemistry 16: 4743–4751. L L, 1972. Fibrinoligase: the stabilising factor system of blood plasma. Ann NY Acad Sci 202: 6–30. L L, C SM, 1984. Transglutaminases. Mol Cell Biol 58: 9–35. L S, D PJA, 1997. Regulation of the tissue transglutaminase gene by DNA methylation. Proc Natl Acad Sci USA 94: 4692–4697. M EJ, T I, C-E R, 1987. Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J Cell Biol 105: 2569–2579. M J, C DG, R RK, S A, B C, 1994. Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers. Biochemistry 33: 2538–2545. M R, B RG, 1993. New members of the collagen superfamily. Curr Opin Cell Biol 5: 883–890. M S, A S, L J, G V, D PAJ, G M, 1995. The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression. FEBS Lett 370: 27–31. M WT, M MP, D PJA, 1984. Retinoic acid-induced expression of tissue transglutaminase in mouse peritoneal macrophages. J Biol Chem 259: 12,794–12,802. N L, T VA, S GL, F L, L W, H RA, C RA, D PJ, 1995. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines. Mol Cell Biol 15: 3540–3551. N H, P DM, B KJ, D T, H A, M K, I MJ, G R, 1994. Gh: a GTP-binding protein
Cell Biology International, Vol. 23, No. 1, 1999
with transglutaminase activity and receptor signalling function. Science 264: 1593–1596. N H, N K, H H, W K, K S, H S, 1991. Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase. Eur J Biochem 202: 15–21. N K, N H, H KI, W S, K S, H S, 1989. Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase. J Biol Chem 264: 19,308–19,312. N M, L TF, 1996. Identification and characterization of up-regulated genes during chondrocyte hypertrophy. Dev Dyn 206: 260–271. O HF, P M, 1990. Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures. Exp Cell Res 191: 292–298. P B, F AM, R M, H D, 1992. Cartilage collagens. In: Adolphe M, ed. Biological regulation of the chondrocytes. Boca Raton, FL, CRC Press. 33–83. P M, C` MP, D. L, D R M, P L, T V, D PJA, F L, 1992. In vivo and in vitro induction of tissue-transglutaminase in rat hepatocytes by retinoic acid. Biochem Biophys Res Commun 1135: 171–179. P RA, 1991. The growth plate: cellular physiology, cartilage assembly and mineralization. In: Hall BK, Newman SA, eds. Cartilage: molecular aspects. Boca Raton, FL, CRC Press. 179–211. R F, D L M, 1990. Complex and divirsified regulatory programs control the expression of vertebrate collagen genes. FASEB J 4: 1616–1623. R AK, D BA, H LA, 1997. Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles. Arthritis Rheum 40: 966–970.
49
S M, G E, P G, A A, D L F, 1991. Expression of type X collagen is transiently stimulated in redifferentiating chondrocytes pretreated with retinoic acid. Biochem J 276: 183–187. S M, P G, A A, G E, 1993. á2(I) collagen gene expression is up-regulated in quail chondrocytes pretreated with retinoic acid. Biochem J 295: 115–119. S M, A A, P G, G E, 1996. The role of cell adhesion in RA-induced modulation of chondrocyte phenotype. Biochem J 313: 201–206. S MM, 1991. Matrix in cartilage and bone development: Current view on the function and regulation of major organic components. Ann Med 23: 207–217. S SM, E G, 1991. Temporal and regional differences in the expression patterns of distinct retinoic acid receptor-â transcripts in the chick embryo. Development 111: 245–252. S T, B PJ, L KN, C E, P MK, 1990. Differential expression of transglutaminase in human erythroleukemia in response to retinoic acid. Cancer Res 50: 7830–7834. S N, I K, S R, 1993. Expression induced by interleukin-6 of tissue type transglutaminase in human hepatoblastoma HepG2 cells. J Biol Chem 268: 7469–7473. R M, G R, 1991. Collagen family of proteins. FASEB J 5: 2814–2823. W-B N, J J-M, M SNP, E JD, L L, 1992. Cloning and expression of chicken erythrocyte transglutaminase. Proc Natl Acad Sci USA 89: 9804–9808. Z L-X, M KJ, D MI, C SJ, J AM, 1995. Evidence for the involvement of RARá-dependent signaling pathway in the induction of tissue transglutaminase and apoptosis by retinoids. J Biol Chem 270: 6022–6029.
TISSUE TRANSGLUTAMINASE EXPRESSION IN QUAIL EPIPHYSEAL CHONDROCYTES ELISA GIONTI1,3*, MASSIMO SANCHEZ2, ANTONIETTA ARCELLA3, GIANFRANCO PONTARELLI3, SIMONA TAVASSI4, VITTORIO GENTILE4, ANNA COZZOLINO4 and RAFFAELE PORTA5 Dipartimento di Medicina Clinica e Sperimentale, Via T. Campanella, 88100 Catanzaro, Italy; 2Istituto Superiore di Sanita`, viale Regina Elena, 299–00161 Roma, Italy; 3Dipartimento di Biochimica e Biotecnologie Mediche, Universita` di Napoli ‘Federico II’, Via S. Pansini, 5–80131 Napoli, Italy; 4Dipartimento di Biochimica e Biofisica, II Universita` di Napoli, Via Costantinopoli, 16–80138 Napoli, Italy; 5CIRPEB Centro Interdipartimentale di Ricerca sui peptidi bioattivi, Universita` di Napoli ‘‘Federico II’’, via Mezzocannone, 4, 80138 Napoli, Italy
1
Received 31 March 1998; accepted 25 September 1998
Tissue transglutaminase (tTGase) is a GTP-binding Ca2+ -dependent enzyme which catalyses the post-translational modification via å(ã-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme. tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation. tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation. Our findings lend support to the idea that tTGase might play a role in non-dividing cultured 1999 Academic Press chondrocytes. K: retinoic acid; tissue transglutaminase; cell adhesion; cell growth; chondrocyte phenotype.
INTRODUCTION Transglutaminases (TGases, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyse post-translational modifications of proteins by the introduction of an isopeptide bond between the ã-carboxamide group of proteinbound glutamines and the å-amino group of protein-bound lysines (for reviews see Folk, 1980; Lorand and Conrad, 1984; Greenberg et al., 1991; Aeschlimann and Paulsson, 1994). Various transglutaminases have been characterized both in *To whom correspondence should be addressed: Dipartimento di Biochimica e Biotecnologie Mediche, Via S. Pansini, 5-80131 Naples, Italy; E-mail: [email protected] 1065–6995/99/010041+09 $30.00/0
intracellular and extracellular compartments (Aeschlimann and Paulsson, 1994). Tissue transglutaminase (tTGase) is a predominantly cytosolic enzyme whose expression is regulated by all transretinoic acid (RA) in vivo and in vitro. Induction of tTGase occurs soon after the addition of RA; the mediators of RA-induced tTGase expression are the nuclear receptors for RA, RAR-á, RAR-â and RAR-ã, as well as the nuclear receptors for 9-cisretinoic acid, RXR’s (Moore et al., 1984; Chiocca et al., 1988; Nara et al., 1989; Piacentini et al., 1992; Nakanishi et al., 1991; Zhang et al., 1995; Nagy et al., 1995). The physiological function of tissue transglutaminase has been associated with the activation of the programmed cell death 1999 Academic Press
42
process (apoptosis) (Fesus et al., 1991; Nagy et al., 1995). The enzyme, however, can also be associated with the cell membrane to exert a receptor-coupled signaling function (Nakaoka et al., 1994; Mian et al., 1995; Chen et al., 1996) or expressed at the extracellular surface of many cells (Barsigian et al., 1991; Martinez et al., 1994). Several extracellular matrix components are tTGase substrates (for review see: Aeschlimann and Paulsson, 1994). In fibroblasts and in endothelial cells, the enzyme plays a role in the extracellular matrix stabilization (Martinez et al., 1994), cell adhesion and cell morphology (Gentile et al., 1992; Jones et al., 1997). The expression of tTGase in cartilage tissues correlates with the terminal differentiation of chondrocytes (Aeschlimann et al., 1993). Cartilage development proceeds through distinct stages starting from mesenchymal cell condensation and resulting in the differentiation of resting chondrocytes. During endochondral ossification, resting chondrocytes undergo a further process of differentiation/maturation involving proliferation, hypertrophy, calcification, degradation and replacement of cartilage by bone and marrow (for reviews see Poole, 1991; Sandberg, 1991; Hunziker, 1994; Erlebacher et al., 1995). In developing long bones tTGase expression correlates with chondrocyte maturation (Aeschlimann et al., 1993). The enzyme is externalized from hypertrophic chondrocytes and might be involved in matrix cross-linking before cartilage undergoes calcification. Furthermore, osteonectin and collagen II are target proteins in hypertrophic chondrocytes (Aeschlimann et al., 1993; Hohenaldl et al., 1995). Extracellular matrix of cartilage contains a variety of proteins including cartilage specific proteoglycan aggrecan, collagen II, tenascin and several minor collagens (Hascall, 1988; van der Rest and Garrone, 1991; Mayne and Brewton, 1993; Mackie et al., 1987). The expression of genes for extracellular matrix components varies during cartilage differentiation/maturation process (for reviews see Poole, 1991; Sandberg, 1991; Hunziker, 1994; Erlebacher et al., 1995). Mesenchymal cells produce collagens I and III; chondrocytes synthesize aggrecan and collagens II, IX, XI and X. The phenotype of cartilage cells undergoes extensive modulation in cell culture as well and the mechanisms regulating the differential expression of extracellular matrix genes are largely unknown (for reviews see Ramirez and Di Liberto, 1990; Petit et al., 1992). Retinoids are a well established means of studying the regulation of extracellular matrix genes in cultured chondrocytes (Benya and Padilla,
Cell Biology International, Vol. 23, No. 1, 1999
1986; Horton et al., 1987; Oettinger and Pacifici, 1990). Primary cultures of quail epiphyseal chondrocytes synthesize the cartilage-specific collagens type II and type IX. Treatment of suspension chondrocytes with retinoic acid inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression (Sanchez et al., 1991, 1993). RA-induced inhibition of the chondrocyte phenotype correlates with cell adhesion. In fact, RA-chondrocytes undergo their phenotypic modulation as they begin to attach. Further, prevention of cell adhesion blocks many of the effects on the chondrocyte phenotype induced by RA (Sanchez et al., 1996). Here we used primary cultures and a permanent cell line of chondrocytes to study tissue transglutaminase expression and its relationships with cell proliferation, cell differentiation and cell adhesion.
MATERIALS AND METHODS Cell culture Primary chondrocytes were isolated from day 10 quail embryo tibiae as previously described (Gionti et al., 1985) with the following modifications. Tibiae were incubated for 1 h in a saline solution containing trypsin/collagenase and the cells released after this incubation are discarded as they consisted mainly of perichondrial cells. Epiphysis were dissected, minced and incubated at 37C in fresh enzyme mixture; cells released by sequential enzymatic digestion from about 50 embryos were pooled and seeded at low initial cell density (2000 cells/cm2). Floating cells were resuspended in Coon’s modified F12 medium (AmbesiImpiombato et al., 1980) supplemented with 10% foetal calf serum. Expression of the differentiated phenotype was assessed by SDS-PAGE of metabolically labeled proteins. all trans-RA (generously provided by Hoffmann-La Roche) was dissolved in 95% ethanol and stored at 80C in the dark. On day 0, this solution was diluted with growth medium and control cultures received an equivalent amount of ethanol. After addition of 0.5 ì RA, cells were plated at 1105 cells/ml. No toxic effect was ever detected with this concentration of RA in treated cultures. All the experiments described in this report were carried out with ordinary foetal calf serum. Parallel experiments in which delipidised serum was used in either control or RA treated cultures did not show significant differences
Cell Biology International, Vol. 23, No. 1, 1999
in tissue transglutaminase activities. To prevent RA-induced cell adhesion, culture dishes were coated with a film of agarose. The continuous cell line MC2 was derived from quail epiphyseal chondrocytes infected with avian myelocitomatosis virus/Rous associated virus-1, subgroup A -MC29(RAV-1)-, a retrovirus carrying the v-myc oncogene (Gionti et al., 1985). Primary epiphyseal chondrocytes can grow either in suspension or in monolayer. Because of their higher sensitivity to viral infection, chondrocytes grown in monolayer were originally used in infection experiments; here, monolayer cells are used as the normal counterpart of the infected cell line. DNA probes The tTGase probe was a full-length clone harbouring a 3200-bp cDNA insert isolated from a cDNA library made from chicken heart (kindly provided by Drs P. Davies and V. Thomazy, Houston, TX, U.S.A.). The coding and the 3 untranslated region shows high homology to the cDNA clone isolated by Weraarchakul-Boonmark et al. (1992). The RAR-â2 probe used was g RAR-â, harbouring a 1600-bp cDNA insert including the entire coding region and the 3 untranslated region of the chicken gene (Smith and Eichele, 1991). The GAPDH probe was a full-length cDNA clone (1200 bp) encoding rat glyceraldehyde-3-phosphate dehydrogenase (Fort et al., 1985). The inserts of these plasmids were used as probes. High specific activity random primed probes were prepared with the Promega kit as specified by the supplier. RNA extraction and Northern blot hybridization Total cellular RNA (2107 cells/sample) extracted as previously described (Chomczynski and Sacchi, 1987), was denatured, fractionated on formaldehyde/agarose gels (Lehrach et al., 1977) and blotted onto an Amersham Hybond N nylon membrane. Pre-hybridization and hybridization were performed in 500 m NaH2PO4 pH 7.2, 7% SDS, 1 m EDTA for 30 min and 16 h, respectively; the hybridization temperature was 65C. Filters were washed three times at 65C in 50 m NaH2PO4 pH 7.2, 1% SDS. Washed filters were dried and exposed to Fuji films with intensifying screens at 80C. Densitometric scanning analysis was performed using software from NIH-Image. Transglutaminase assay Cells (3106 cells/sample) were mechanically removed from dishes, rinsed twice with PBS, resus-
43
pended and homogenized in Tris–HCl 20 m (pH 7.4) containing 2 m EGTA, pepstatin (0.1 ìg/ 100 ìl), leupeptin (0.1 ìg/100 ìl), PMSF 0.1 m, bestatin (0.1 ìg/100 ìl) and sonicated at 4C for 20 s. Transglutaminase activity was measured by detecting the incorporation of [3H]spermidine into N,N -dimethylcasein as previously reported (Lorand, 1972). The incubation mixture contained 125 m Tris–HCl (pH 8.0), 2.5 m CaCl2, 10 m dithiothreitol (DTT), 200 ìg N,N -dimethylcasein, 12.5 ì spermidine containing 0.15 ìCi [3H]spermidine and 5 to 25 ìg of protein from total cell homogenate in a final volume of 0.1 ml. After 30 min incubation at 37C, the samples were precipitated with 10% TCA. Free [3H]spermidine was eliminated by washing with large volumes of cold 10% TCA containing 1 m spermidine. The pellet was resuspended and bound [3H]spermidine was counted with 5 ml of Pico Fluor 30 scintillation cocktail (United Technologies/Packard). Chondrocytes enzyme activity was inhibited by adding EGTA thus suggesting that it is calciumdependent. Western blot analysis Determination of tissue transglutaminase protein was carried out with condrocytes lysates by Western blot analysis. tTGase-positive bands were revealed by using as a primary antibody an affinity purified IgG raised in goat against purified guinea pig liver tTGase proven to be not cross-reactive with other forms of transglutaminase (Chiocca et al., 1988). Statistical analysis Statistical significance of tTGase mRNA levels between control and RA-treated chondrocytes were calculated using Student’s t-test. RESULTS Retinoic acid stimulates tTGase expression in primary chondrocytes Like chondrocytes from many species, quail epiphyseal chondrocytes (QEC) can grow either in monolayer as polygonal cells or in suspension. Since suspension quail chondrocytes convert poorly into attached cells, they are a suitable system in which to study the alterations in cellmatrix interactions that occur during RA-induced changes in cell shape (Sanchez et al., 1991).
44
Cell Biology International, Vol. 23, No. 1, 1999
Fig. 1. Morphology of control and RA treated primary chondrocytes. A: control untreated chondrocytes (the picture was taken 3 days after plating); B, C and D: chondrocytes treated with 0.5 ì RA for 1, 2 and 3 days, respectively. Cells were photographed under phase contrast: magnification 75.
Exposure of suspension QEC to RA for three days blocks cell growth, inhibits the expression of cartilage-specific collagens type II and IX, turns on fibronectin expression and induces cell adhesion (Sanchez et al., 1991, 1996). One day after RA addition about 50% of the cells adhere to the substrate; by 2 days of treatment adhesion is almost complete (Fig. 1). By 3 days many cells display a dedifferentiated flattened morphology consistent with the results obtained by Hassel et al. (1979). Transglutaminase activity was found in chondrocyte lysates (Table 1). Enzyme activity increases upon RA treatment and reaches its maximum after around 16 h of treatment then gradually declines to slightly lower values than the base-line activity detected in control chondrocytes. Stimulation of enzyme activity is accompanied by parallel and equivalent increases of a 77-kDa band as detected by Western blot analysis in cell lysates from control and RA treated chondrocytes (not shown). A single 3.2-kb band was detected by hybridization of total RNA from suspension QEC with a cDNA clone encoding chicken tissue-transglutaminase (Fig. 2). In the experiment shown in Figure 2, stimulation of tTGase expression occurs between 4 and 8 h after RA addition and remains unchanged over the subsequent 16 h. Thereafter, tTGase mRNA levels decline until at 72 h they reach the basal levels detected in untreated control chondrocytes (the results are not shown but have been made available
to the referees). RAR-â2 expression is readily induced upon RA addition (Fig. 2), consistently with the results obtained in chicken sternal chondrocytes by Iwamoto et al. (1993a). RA-treated chondrocytes display a 2–4-fold increase of tTGase mRNA over the levels detected in untreated control chondrocytes as evaluated in different experiments by densitometric scanning analysis and normalization to GAPDH expression (Table 2). Cell adhesion does not correlate with tTGase expression in untreated chondrocytes: similar levels of tTGase expression were observed in Table 1. Transglutaminase activity in chondrocyte homogenates from primary cultures treated with 0.5 ì RA for the indicated times Treatment (h)
Control 8 16 24 40 72
Transglutaminase activity* (pmoles of [3H]spermidine incorporated/mg protein) 26.00.49 45.50.88 66.91.37 52.31.21 43.80.71 15.61.4
*Values represent the mean value of three different experiments carried out by triplicate determinations.. Further experimental details are reported in the Materials and Methods.
Cell Biology International, Vol. 23, No. 1, 1999
45
Fig. 2. Time course of RA-induced stimulation of tissue transglutaminase and RAR-â2 genes in primary cultures of quail epiphyseal chondrocytes. Steady-state levels of tTGase, RAR-â2 and GAPDH mRNAs in control chondrocytes (lane 1) and in chondrocytes treated with 0.5 ì RA for 4, 8 and 24 h (lanes 2, 3 and 4, respectively).
untreated cultures growing either in suspension or in monolayer as judged by Northern blot (Fig. 2, lane 1; Fig. 3, lane 1), by Western blot analysis and by enzyme activity assays (not shown). To investigate whether the stimulation of tTGase expression correlates with RA-induced cell Table 2. Relative levels of tTGase mRNA (fold of stimulation) in RA-treated chondrocytes
Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7 Means ..
Monolayer
Suspension
3.5 1.6 3.45 4.4 — — 3 3.19** 1.023719
2.75 — — 2.25 4.8 2.75 — 3.1375* 1.133119
Primary chondrocytes were cultured either in permissive conditions for RA-induced cell adhesion (Monolayer) or in non-permissive conditions for RA-induced cell adhesion (Suspension). Total RNAs extracted from control and RA-treated chondrocytes were isolated and analysed by Northern blot for tTGase expression. tTGase and GAPDH bands were scanned and the scan units were converted into -fold stimulation over the basal levels detected in control chondrocytes. In the experiments 1, 3, 6, the reported values refer to stimulations obtained after 6 h of RA-treatment; in the other experiments (2, 4, 5, 7) the reported values refer to shorter treatments (4 h). Significant differences from controls are designated as *P<0.05, **P<0.01. —, Not done.
Fig. 3. Tissue transglutaminase responsiveness to RA treatment in primary chondrocytes and MC2, a chondrocyte cell line immortalized by the activated form of Myc. Steady-state mRNA levels of tTGase and GAPDH mRNAs in untreated primary chondrocytes grown in monolayer (lane 1); primary chondrocytes treated with RA for 8 h (lane 2); in untreated MC2 cells (lane 3) and MC2 cells treated with RA for 8 h and 24 h (lanes 4 and 5, respectively). Because of their higher sensitivity to viral infection, primary chondrocytes grown in monolayer have been originally used in the infection experiments; here they are used as the normal counterpart of the infected cell line.
adhesion, we prevented cell adhesion by culturing chondrocytes on agarose-coated dishes. We have previously shown that RA treatment does not significantly affect the expression of the chondrocyte phenotype if cell adhesion is prevented (Sanchez et al., 1996). Stimulation of tTGase expression occurs in non permissive conditions for RA-induced cell adhesion (Table 2), while the RA treatment fails to inhibit the chondrocyte phenotype as assessed by SDS-PAGE of metabolically labeled proteins (not shown). Equivalent increases of enzyme activity and tTGase protein levels also occur (not shown). RA treatment blocks cell proliferation both in permissive and in non permissive conditions for cell adhesion (Sanchez et al., 1996). Cell survival was assayed at the end of RA treatment: most of the cells (90%) retained the ability to exclude Trypan Blue, regardlesss of culture conditions. tTGase expression in MC2, a cell line derived from retrovirus infected chondrocytes We next analysed the tTGase expression in a chondrocyte cell line we have established in our laboratory from primary QEC infected with avian myelocytomatosis virus -MC29 (RAV-1)-, a v-myccarrying retrovirus (Gionti et al. 1985). Infection of monolayer QEC with MC29 (RAV-1) does not induce cell transformation, stimulates cell proliferation and leads to the establishment of a continuous cell line, designated as MC2, which maintains the expression of cartilage differentiation markers besides the typical polygonal morphology of chondrocytes growing in monolayer (Gionti et al., 1985).
46
Differently from uninfected chondrocytes, we could not observe tTGase transcripts in MC2 cells and RA treatment fails to activate tTGase expression in these cells as assessed by Northern blot (Fig. 3). Prolonged exposure to RA has no significant effect on tTGase expression (not shown). The rate of proliferation of MC2 cells is 50% inhibited after a 3d treatment. RAR-â2 expression is readily induced by RA treatment in MC2 cells as assessed by Northern blot analysis (not shown). DISCUSSION Here we report that tTGase activity is present in primary cultures of quail epiphyseal chondrocytes (QEC) and increases transiently after RA treatment. Increase in steady-state levels of tTGase mRNA occurs between 4 and 8 h of RA treatment. Conversely, the inhibition of cartilage phenotypic markers and changes in cell morphology we observe in RA treated cultures become apparent only after several hours or even days of exposure to RA (Sanchez et al. 1991, 1993, 1996). Such delayed responses likely result from changes in gene expression secondary to the direct action of RA and its receptors. In vivo, tissue transglutaminase distribution correlates with chondrocyte maturation (Aeschlimann et al. 1993). Chondrocyte maturation is the complex developmental process starting after chondrocyte differentiation and resulting in mature hypertrophic chondrocytes which produce an extracellular matrix permissive for calcification (for reviews see: Poole, 1991; Hunziker, 1994). In permanent cartilage, e.g. normal articular cartilage, chondrocyte maturation does not take place but it is interrupted at the stage of resting chondrocytes. tTGase activity is accumulated intracellularly in the proliferation/maturation zone and it is externalized by hypertrophic chondrocytes before cartilage undergoes calcification (Aeschlimann et al., 1993). The transglutaminase reaction products, the ã-glutamyl-å-lysine cross-links, are abundant in the mineralizing cartilage matrix during the endochondral bone formation and the maturation of cartilages that do not undergo ossification, e.g., tracheal and laryngeal cartilages (Aeschlimann et al., 1995). Osteonectin and collagen II are target proteins for cross-linking in hypertrophic chondrocytes (Aeschlimann et al., 1995; Hohenaldl et al., 1995). The enzyme responsible for cross-linking of the mineralizing cartilage matrix is tTGase while it remains unclear which transglutaminase is expressed in osteoblasts (Aeschlimann et al. 1996).
Cell Biology International, Vol. 23, No. 1, 1999
Numirskaya and Linsenmayer (1996) identified in cultured hypertrophic chondrocytes a chicken homologue of the human Factor XIIIa which was upregulated 3–4-fold over non-hypertrophic chondrocytes. These findings are not consistent with the immunohistochemical identification of the cartilage molecule as tTGase (Aeschlimann et al. 1993). Perhaps this discrepancy was due to aspecific activation of gene expression produced by the in vitro environment. Alternatively, hypertrophic chondrocytes may express more than one form of transglutaminase. Two forms of transglutaminase activity that differed in their cellular localization were reported in cultured articular chondrocytes from rabbits (Demignot et al., 1995) and from adult pigs (Rosenthal et al., 1997) while Aeschlimann et al. (1993) found no TGase activity in articular cartilages from rats and calves. Rabbit articular chondrocytes readily dedifferentiate upon subculture (Benya and Padilla, 1986); similarly, transglutaminase activity drops at the second passage. TGase activity in rabbit articular chondrocytes was mainly membrane associated and was downregulated by RA treatment (Demignot et al., 1995) while the cytosolic activity was identified as tTGase (Borge et al., 1996). The present report dealing with tTGase activity in primary QEC, which display a differentiated phenotype upon subculture, provides further support to the idea that tTGase activity has a physiological role in cartilage cells. Upregulation of tTGase by RA is well established (Moore et al., 1984; Chiocca et al., 1988; Nara et al., 1989; Piacentini et al., 1992; Gentile et al., 1992) and RA has been shown to play an important role in the regulation of chondrocyte maturation and cartilage mineralization (Iwamoto et al., 1993b; Ballock et al., 1994). RA-induced inhibition of the differentiation programme in suspension QEC is associated with growth arrest and induction of cell adhesion (Sanchez et al., 1991, 1996). Increased enzyme activity might lead to cross-linking of extracellular matrix proteins thus contributing to matrix remodeling that occurs upon RA-induced cell adhesion. It has been suggested that in other cell types increased tTGase activity correlates with morphological changes (Byrd and Licthi, 1987; Nara et al., 1989: Nakanishi et al., 1991) and increased cell adhesion (Cai et al., 1991; Gentile et al., 1992; Jones et al., 1997). In quail epiphyseal chondrocytes, RAinduced cell adhesion correlates with the inhibition of the expression of chondrocyte phenotypic traits. In fact, we have observed that prevention of cell
Cell Biology International, Vol. 23, No. 1, 1999
adhesion results in the failure of the RA-induced inhibition of the chondrocyte phenotype (Sanchez et al., 1996). In culture conditions non-permissive for cell adhesion we still observe RA-induced stimulation of tTGase expression. Therefore, stimulation of tTGase by RA can be uncoupled from the inhibition of the chondrocyte phenotype by RA which, in contrast, is dependent on cell adhesion and cell shape changes. These results suggest that stimulation of tTGase expression in QEC might result from a more direct effect of RA and its receptors on the regulation of this gene rather than being subsequent to cell adhesion. Borge et al. (1996) showed that tTGase expression in rabbit articular chondrocytes correlates with cell adhesion and spreading regardless of their differentiation state; conversely, in quail epihyseal chondrocytes tTGase gene is expressed in both suspension and monolayer cultures and stimulation by RA occurs independently of cell adhesion. In considering these discrepancies, it must be noted that there are several important variations in the study we now report. In addition to the differences in the species and origin of the cells, dedifferentiation of rabbit articular chondrocytes starts as soon as the first passage (Demignot et al., 1995), while QEC display a differentiated phenotype upon prolonged culture (Sanchez et al., 1996). Whether these discrepancies relate to the maturation state of the responding cells in these two culture systems or they pertain to some other phenomena, remains to be determined. Our data show that RA-induced stimulation of tTGase expression in primary cultures of chondrocytes correlates with cell growth suppression. It has been reported that inhibition of transglutaminase activity results in the stimulation of cell proliferation in WI-38, a lung cell line (Birckbichler et al., 1981). Further, reduced levels of transglutaminase have been observed in tumors (Barnes et al., 1985; Knight et al., 1991) and in transformed cells (Birckbichler et al., 1977; Kosa et al., 1993). Here we report the loss of tTGase expression in a continuously growing chondrocyte cell line and an increased gene expression in growth arrested primary chondrocytes. It has been proposed that tTGase activity might play a role in the control of cell growth (Birckbichler et al., 1981; Mian et al., 1995). In this regard, our observations support this hypothesis. Lu and Davies (1997) have shown that alterations in DNA methylation of tTGase promoter may be one of the mechanisms regulating the expression of this gene. Whether the loss of tTGase expression in the chondrocyte cell line correlates with hypermethylation of the tTGase promoter
47
or with some other mechanisms remains to be elucidated. An understanding of the biological role of tTGase in cartilage cells will further our understanding of the complex molecular mechanisms underlying the control of cell growth and differentiation in this cell lineage.
ACKNOWLEDGEMENTS We are much indebted with Dr P. J. A. Davies and Dr V. Thomazy (Dept. of Pharmacology, Medical School, University of Texas, Houston) for providing us with the cDNA clone for chicken tTGase used in this study. Dr P. J. A. Davies has also provided the tTGase antibody. The financial support from MURST 40% ‘Meccanismi di Cancerogenesi’, PRIN ‘Struttura e regolazione dell’espressione dei geni in eucarioti’ and TelethonItaly grant #E738 is gratefully acknowledged. We thank V. Brescia for technical assistance. REFERENCES A D, W A, F H, P M, 1993. Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation in condrocytes. J Cell Biol 120: 1461–1470. A D, P M, 1994. Transglutaminases: protein cross-linking enzymes in tissue and body fluids. Thromb Haemost 71: 402–415. A D, K O, P M, 1995. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J Cell Biol 129: 881–892. A D, M D, P M, 1996. Tissuetransglutaminase and Factor XIII in cartilage and bone remodeling. Semin Thromb Hemost 22: 437–443. A-I FS, P LAM, C HG, 1980. Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA 77: 3455–3459. B RT, H A, W LM, F KC, R AB, S MB, 1994. Inhibition of chondrocyte phenotype by retinoic acid involves up-regulation of metalloprotease genes independent of TGF-â. J Cell Physiol 159: 340–346. B RN, B PJ, E BM, W PL, G M, 1985. Alterations in the distribution and activity of transglutaminase during tumor growth and metastasis. Carcinogenesis 6: 459–463. B C, S AM, M J, 1991. Tissue transglutaminase covalently incorporates itself, fibrinogen or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. J Biol Chem 266: 22,501–22,509. B PD, P SR, 1986. Modulation of rabbit chondrocyte phenotype by retinoic acid terminates type II collagen
48
synthesis without inducing type I collagen: the modulated phenotype differs from that produced by subculture. Dev Biol 112: 296–305. B PJ, O GR, C E, P MK, 1977. Transglutaminase activity in normal and transformed cells. Cancer Res 37: 1340–1344. B PJ, O GR, P MK, C E, C HA, 1981. Increase in proliferative markers after inhibition of transglutaminase. Proc Natl Acad Sci USA 78: 5005–5008. B L, D S, A M, 1996. Type II transglutaminase expression in rabbit articular chondrocytes in culture: relation with cell differentiation, cell growth, cell adhesion and cell apoptosis. Biochim Biophys Acta 1312: 117–124. B JC, L U, 1987. Two types of transglutaminase in PC12 pheochromocytoma cell line. J Biol Chem 262: 11,699–11,705. C D, B T, D L LM, 1991. Retinoids induce transglutaminase in NIH 3T3 cells. Biochem Biophys Res Commun 175: 1119–1124. C S, L F, I S, L KN, B PJ, G RM, 1996. á1-adrenergic receptor signaling via Gh is subtype specific and independent of its transglutaminase activity. J Biol Chem 271: 32,385–32,391. C EA, D PJA, S JP, 1988. The molecular basis of retinoic acid action: transcriptional regulation of tissue transglutaminase gene expression in macrophage. J Biol Chem 263: 11,584–11,589. C P, S N, 1987. Single step method of RNA isolation by acid guanidium thiocianate-phenol-chloroform extraction. Anal Biochem 162: 156–159. D S, B L, A M, 1995. Transglutaminase activity in rabbit articular chondrocytes in culture. Biochim Biophys Acta 1266: 163–170. E A, F EH, G SE, D R, 1995. Toward a molecular understanding of skeletal development. Cell 80: 371–378. F L, D PJA, P M, 1991. Apoptosis: molecular mechanisms in programmed cell death. Eur J Cell Biol 56: 170–177. F JE, 1980. Transglutaminases. Annu Rev Biochem 49: 517–531. F P, M L, P M, S SE, D C, J P, B JM, 1985. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigenic family. Nucl Acid Res 13: 1431–1442. G V, T V, P M, F L, D PJA, 1992. Expression of tissue transglutaminase in Balb-c 3T3 fibroblasts: Effects on cellular morphology and adhesion. J Cell Biol 119: 463–474. G E, P G, C R, 1985. Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes. Proc Natl Acad Sci USA 82: 2756–2760. G CS, B PJ, R RH, 1991. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J 5: 3071–3077. H VC, 1988. Proteoglycans: the chondroitin sulfate/ keratan sulfate proteoglycan of cartilage. ISI Atlas Sci (Biochem): 189–198. H JR, P JP, K HK, P RM, Y KM, 1979. Enhanced cellular fibronectin accumulation in chondrocytes treated with vitamin A. Cell 17: 821–826.
Cell Biology International, Vol. 23, No. 1, 1999
H C, M K, M U, T R, P M, A D, 1995. Two adjacent N-terminal glutamines of BM-40 (Osteonectin-SPARC) act as amine acceptor sites in transglutaminasec-catalyzed modification. J Biol Chem 270: 23,415–23,420. H WE, Y Y, H JR, 1987. Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes. Dev Biol 123: 508–516. H EB, 1994. Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Technique 28: 505–519. I M, G EB, A SL, N S, P M, 1993a. Responsiveness to retinoic acid changes during chondrocyte maturation. Exp Cell Res 205: 213–224. I M, S IM, Y K, B AL, L PS, A, SL, P M, 1993b. Retinoic acid induces rapid mineralization and expression of mineralization-related genes in chondrocytes. Exp Cell Res 207: 413–420. J RA, N B, M S, D PJA, G M, 1997. Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin. J Cell Sci 110: 2461–2472. K CRL, R RC, G M, 1991. Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumor progression. Biochem Biophys Acta 1096: 312–318. K K, M K, D L LM, 1993. Transformation of NIH3T3 cells with ras oncogenes abrogates the retinoic acid induction of tissue transglutaminase. Biochem Biophys Res Commun 196: 1025–1033. L H, D D, W J, B H, 1977. RNA molecular weight determination by gel electrophoresis under denaturing conditions. A critical reexamination. Biochemistry 16: 4743–4751. L L, 1972. Fibrinoligase: the stabilising factor system of blood plasma. Ann NY Acad Sci 202: 6–30. L L, C SM, 1984. Transglutaminases. Mol Cell Biol 58: 9–35. L S, D PJA, 1997. Regulation of the tissue transglutaminase gene by DNA methylation. Proc Natl Acad Sci USA 94: 4692–4697. M EJ, T I, C-E R, 1987. Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J Cell Biol 105: 2569–2579. M J, C DG, R RK, S A, B C, 1994. Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers. Biochemistry 33: 2538–2545. M R, B RG, 1993. New members of the collagen superfamily. Curr Opin Cell Biol 5: 883–890. M S, A S, L J, G V, D PAJ, G M, 1995. The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression. FEBS Lett 370: 27–31. M WT, M MP, D PJA, 1984. Retinoic acid-induced expression of tissue transglutaminase in mouse peritoneal macrophages. J Biol Chem 259: 12,794–12,802. N L, T VA, S GL, F L, L W, H RA, C RA, D PJ, 1995. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines. Mol Cell Biol 15: 3540–3551. N H, P DM, B KJ, D T, H A, M K, I MJ, G R, 1994. Gh: a GTP-binding protein
Cell Biology International, Vol. 23, No. 1, 1999
with transglutaminase activity and receptor signalling function. Science 264: 1593–1596. N H, N K, H H, W K, K S, H S, 1991. Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase. Eur J Biochem 202: 15–21. N K, N H, H KI, W S, K S, H S, 1989. Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase. J Biol Chem 264: 19,308–19,312. N M, L TF, 1996. Identification and characterization of up-regulated genes during chondrocyte hypertrophy. Dev Dyn 206: 260–271. O HF, P M, 1990. Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures. Exp Cell Res 191: 292–298. P B, F AM, R M, H D, 1992. Cartilage collagens. In: Adolphe M, ed. Biological regulation of the chondrocytes. Boca Raton, FL, CRC Press. 33–83. P M, C` MP, D. L, D R M, P L, T V, D PJA, F L, 1992. In vivo and in vitro induction of tissue-transglutaminase in rat hepatocytes by retinoic acid. Biochem Biophys Res Commun 1135: 171–179. P RA, 1991. The growth plate: cellular physiology, cartilage assembly and mineralization. In: Hall BK, Newman SA, eds. Cartilage: molecular aspects. Boca Raton, FL, CRC Press. 179–211. R F, D L M, 1990. Complex and divirsified regulatory programs control the expression of vertebrate collagen genes. FASEB J 4: 1616–1623. R AK, D BA, H LA, 1997. Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles. Arthritis Rheum 40: 966–970.
49
S M, G E, P G, A A, D L F, 1991. Expression of type X collagen is transiently stimulated in redifferentiating chondrocytes pretreated with retinoic acid. Biochem J 276: 183–187. S M, P G, A A, G E, 1993. á2(I) collagen gene expression is up-regulated in quail chondrocytes pretreated with retinoic acid. Biochem J 295: 115–119. S M, A A, P G, G E, 1996. The role of cell adhesion in RA-induced modulation of chondrocyte phenotype. Biochem J 313: 201–206. S MM, 1991. Matrix in cartilage and bone development: Current view on the function and regulation of major organic components. Ann Med 23: 207–217. S SM, E G, 1991. Temporal and regional differences in the expression patterns of distinct retinoic acid receptor-â transcripts in the chick embryo. Development 111: 245–252. S T, B PJ, L KN, C E, P MK, 1990. Differential expression of transglutaminase in human erythroleukemia in response to retinoic acid. Cancer Res 50: 7830–7834. S N, I K, S R, 1993. Expression induced by interleukin-6 of tissue type transglutaminase in human hepatoblastoma HepG2 cells. J Biol Chem 268: 7469–7473. R M, G R, 1991. Collagen family of proteins. FASEB J 5: 2814–2823. W-B N, J J-M, M SNP, E JD, L L, 1992. Cloning and expression of chicken erythrocyte transglutaminase. Proc Natl Acad Sci USA 89: 9804–9808. Z L-X, M KJ, D MI, C SJ, J AM, 1995. Evidence for the involvement of RARá-dependent signaling pathway in the induction of tissue transglutaminase and apoptosis by retinoids. J Biol Chem 270: 6022–6029.