TGF-BETA-1 REGULATION OF COLLAGEN-METABOLISM BY EMBRYONIC PALATE MESENCHYMAL CELLS

Proper metabolism of the extracellular matrix (ECM) in mammalian embry onic palatal tissue is required for normal development of the palate. In particular, perturbation of collagen metabolism in the embryonic or ofacial region results in the production of cleft palate. Although sev eral types of collagen have been localized in the embryonic palate, fa ctors responsible for regulating their synthesis have not been identif ied. Transforming growth factor beta (TGF beta), shown to be capable o f modulating ECM metabolism in other tissues, has been localized in th e developing palate. Thus, we examined the ability of TGF beta 1 to mo dulate collagen synthesis and degradation in murine embryonic palate m esenchymal (MEPM) cells in vitro. Immunohistochemical analysis confirm ed that type III collagen was predominant in the mesenchyme of the emb ryonic palate, whereas type I collagen was ubiquitous throughout palat al epithelium and mesenchyme. Total collagen production by TGF beta-tr eated confluent MEPM cells in serum-free conditioned medium was determ ined by measuring incorporation of L-[2-3-4-5-H-3]proline into hydroxy proline. Treatment for 24 hr with TGF beta 1 stimulated incorporation into both cell layer and medium fractions. Quantification of collagen types by ELISA indicated that TGF beta 1 stimulated the accumulation o f type III collagen as early as 3 hr after treatment. Northern blot an alysis of MEPM cells treated with TGF beta 1 revealed that steady-stat e levels of mRNA encoding for procollagen alpha 1(I) and alpha 1(III) were increased and that these effects were ablated by cycloheximide bu t not actinomycin. The effects of TGF beta treatment on MEPM cell coll agen levels also reflected alterations in collagen degradation. TGF be ta-treated MEPM cells exhibited a significant diminution of total prot ease activity. Moreover, analysis by substrate gel electrophoresis ind icated specific decreases in vertebrate collagenase and stromelysin. T hese data represent the first report of changing proteolytic profiles during palatogenesis. Thus, TGF beta regulates the amount of collagen present in embryonic palatal tissue at the level of synthesis and degr adation. (C) 1994 Wiley-Liss, Inc.