Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization

Abstract

During the process of endochondral bone formation, chondrocytes undergo a series of complex maturational changes. Our recent studies indicate that this maturational process is influenced by the vitamin A derivative retinoic acid (RA). To learn how this agent regulates chondrocyte development, we characterized matrix gene expression during maturation of cartilage cells in chick sternum. RNAs were isolated from the cephalic portion of day 13, 14, 16, 18, and 20 chick embryo sternum and analyzed via northern blots. Type II collagen RNA levels remained fairly constant during this developmental period. In contrast, expression of type X collagen and alkaline phosphatase (APase) genes was first detected at day 16, followed by that of osteonectin (ON) and osteopontin (OP). To explore the mechanisms triggering these changes, chondrocytes were isolated from the cephalic portion of day 17-18 sternum (US cells) and grown in monolayer in standard serum-containing medium. After 3 weeks in culture, most of the cells enlarged and became type X collagen-positive, but they exhibited low APase activity and contained only trace amounts of ON and OP mRNAs. Treatment of parallel 3-week-old cultures with RA (10-100 nM) rapidly increased expression of the APase, ON, and OP genes severalfold. In concert with a significant increase in APase activity, there was abundant calcium accumulation in the RA-treated cultures. Electron microscopy confirmed the formation of large matrix-associated mineral crystals and the presence of numerous matrix vesicles. The effects of RA were also studied in cultures of immature chondrocytes isolated from the caudal portion of sternum (LS cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Cartilage Calcification Studied by Proton Nuclear Magnetic Resonance Microscopy

“…The cells were terminally differentiated with retinoic acid, which induces late maturation genes and activates the mineralization of the cartilage matrix. (17,18) Ascorbic acid and …”

“…(17,18) After retinoic acid treatment, the tissue culture medium was supplemented with 1% ␤-glycerophosphate (Sigma) and 50 g/ml of ascorbic acid to facilitate mineral formation.…”

The promoter of the mouse tissue transglutaminase gene directs tissue-specific, retinoid-regulated and apoptosis-linked expression

“…This may explain why the transglutaminase transgene is appropriately expressed in tissues such as cartilage and the limb mesenchyme where retinoids have been implicated in the physiological control of gene expression (Helms et al, 1996;Kastner et al, 1995;Kochhar et al, 1993;Ywamoto et al, 1994). These sites are not simply sites of generic retinoid-regulated gene expression however since a different retinoid regulated reporter transgene, the RARb-lacZ transgene, shows different sites of expression during limb development in the mouse (Mendelsohn et al, 1991).…”

“…Studies from a number of laboratories have shown that retinoids play a critical role in regulating chondrogenic differentiation and particularly the patterns of gene expression of hypertrophic chondrocytes (Cash et al, 1997;Oettinger and Pacifici, 1990;Pacifici et al, 1991;Ywamoto et al, 1993Ywamoto et al, , 1994. Retinoids also induce tissue transglutaminase expression in cultured chondrocytes (V. Gentile personal comm.).…”

Distinct Transglutaminase 2-independent and Transglutaminase 2-dependent Pathways Mediate Articular Chondrocyte Hypertrophy

“…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).…”