DIFFERENTIAL EXPRESSION AND BIOLOGICAL-ACTIVITY OF RETINOIC ACID-INDUCED TGF-BETA ISOFORMS IN EMBRYONIC PALATE MESENCHYMAL CELLS

The effect of retinoic acid (RA) on TGF-beta mRNA expression and prote in production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-beta bioassay in association wit h TGF-beta isoform-specific neutralizing antibodies. Heat or acid acti vation was used to distinguish between latent and active TGF-beta prot ein released into the culture medium. RA had little or no effect on TG F-beta 1 mRNA expression and protein production. In contrast, RA incre ased TGF-beta 2 and beta 3 protein released into the culture medium, t he protein being mostly in an inactive or latent form. The amount of a ctive TGF-beta released was increased relative to the total increase i n TGF-beta released, suggesting that RA treatment stimulated activatio n of latent TGF-beta. RA also increased TGF-beta 2 mRNA expression; we have previously shown that RA upregulates TGF-beta 3 mRNA in these ce lls. RA and TGF-beta individually inhibited H-3-thymidine incorporatio n into MEPM cell DNA, while, when administered simultaneously, they in hibited proliferative activity to a greater extent. Heat- or acid-acti vated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit H-3-thymidine incorporation into MEPM cell DNA to an exten t greater than seen with RA treatment alone. Coincubation of heat-acti vated CM from RA-treated MEPM cells with pan-specific or TGF-beta 2 or beta 3-specific neutralizing antibodies partially relieved the inhibi tory effect on H-3-thymidine incorporation, suggesting that this proli ferative response was due to RA-induced TGF-beta. Simultaneous treatme nt with RA and TGF-beta also stimulated gycosaminoglycan (GAG) synthes is to an extent greater than that seen with TGF-beta treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data de monstrate a role for RA and RA-induced TGF-beta in the regulation of p alate cell proliferation and GAG synthesis and suggest a role for TGF- beta in retinoid-induced cleft palate. J. Cell. Physiol. 177:36-46, 19 98. (C) 1998 Wiley-Liss, Inc.