High‐dose retinoic acid modulates rat calvarial osteoblast biology

Abstract

Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.

Retinoic acid-induced premature osteoblast-to-preosteocyte transitioning has multiple effects on calvarial development

“…In fact, in vitro studies have provided evidence for both inhibitory and stimulatory effects of RA on both osteoclast (Balkan et al, 2011;Chiba et al, 1996) and osteoblast (Cohen-Tanugi and Forest, 1998;Iba et al, 2001;Skillington et al, 2002;Song et al, 2005) differentiation, depending on the culture conditions and the cell lines used.…”

Osteoblast de- and redifferentiation is controlled by a dynamic response to retinoic acid during zebrafish fin regeneration

“…Retinoic acid (RA) has been shown to play multiple roles in bone development and repair by exerting pleiotropic effects on cells of the chondroblast, osteoblast and osteoclast lineages (Adams et al, 2007;Allen et al, 2002;Conaway et al, 2013;Dranse et al, 2011;Koyama et al, 1999;Laue et al, 2008Laue et al, , 2011Li et al, 2010;Lie and Moren, 2012;Lind et al, 2013;Nallamshetty et al, 2013;Song et al, 2005;Spoorendonk et al, 2008;Weston et al, 2003;Williams et al, 2009). Although partially conflicting results were reported, the general consensus for osteoblastogenesis is that RA signaling restricts osteoblast differentiation but promotes subsequent bone matrix synthesis by mature osteoblasts.…”

Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton

“…Some studies describe an increase in mineralization (Skillington et al, 2002;Wang and Kirsch, 2002;Song et al, 2005;Yamashita et al, 2005;Malladi et al, 2006;Wan et al, 2007), whereas others report a suppression of cell differentiation and therefore a decrease in mineralization (Cohen-Tanugi and Forest, 1998;Iba et al, 2001) upon RA treatment of cultured cells. Here, we establish an in vivo model to follow early osteoblasts.…”