TGFβ1 regulation of collagen metabolism by embryonic palate mesenchymal cells

Abstract

Proper metabolism of the extracellular matrix (ECM) in mammalian embryonic palatal tissue is required for normal development of the palate. In particular, perturbation of collagen metabolism in the embryonic orofacial region results in the production of cleft palate. Although several types of collagen have been localized in the embryonic palate, factors responsible for regulating their synthesis have not been identified. Transforming growth factor β (TGFβ), shown to be capable of modulating ECM metabolism in other tissues, has been localized in the developing palate. Thus, we examined the ability of TGFβ1 to modulate collagen synthesis and degradation in murine embryonic palate mesenchymal (MEPM) cells in vitro. Immunohistochemical analysis confirmed that type III collagen was predominant in the mesenchyme of the embryonic palate, whereas type I collagen was ubiquitous throughout palatal epithelium and mesenchyme. Total collagen production by TGFβ-treated confluent MEPM cells in serum-free conditioned medium was determined by measuring incorporation of L-[2-3-4-5-³H]-proline into hydroxyproline. Treatment for 24 hr with TGFβ1 stimulated incorporation into both cell layer and medium fractions. Quantification of collagen types by ELISA indicated that TGFβ1 stimulated the accumulation of type III collagen as early as 3 hr after treatment. Northern blot analysis of MEPM cells treated with TGFβ1 revealed that steady-state levels of mRNA encoding for procollagen α1(I) and α1(III) were increased and that these effects were ablated by cycloheximide but not actinomycin. The effects of TGFβ treatment on MEPM cell collagen levels also reflected alterations in collagen degradation. TGFβ-treated MEPM cells exhibited a significant diminution of total protease activity. Moreover, analysis by substrate gel electrophoresis indicated specific decreases in vertebrate collagenase and stromelysin. These data represent the first report of changing proteolytic profiles during palatogenesis. Thus, TGFβ regulates the amount of collagen present in embryonic palatal tissue at the level of synthesis and degradation.