D'Angelo M, Chen J M, Ugen K, Greene R M
Department of Anatomy, Pathology and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Exp Zool. 1994 Oct 1;270(2):189-201. doi: 10.1002/jez.1402700208.
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 beta (TGF beta), shown to be capable of modulating ECM metabolism in other tissues, has been localized in the developing palate. Thus, we examined the ability of TGF beta 1 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 TGF beta-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 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 of type III collagen as early as 3 hr after treatment. Northern blot analysis of MEPM cells treated with TGF beta 1 revealed that steady-state levels of mRNA encoding for procollagen alpha 1 (I) and alpha 1 (III) were increased and that these effects were ablated by cycloheximide but not actinomycin. The effects of TGF beta treatment on MEPM cell collagen levels also reflected alterations in collagen degradation. TGF beta-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, TGF beta regulates the amount of collagen present in embryonic palatal tissue at the level of synthesis and degradation.
哺乳动物胚胎腭组织中细胞外基质（ECM）的正常代谢是腭正常发育所必需的。特别是，胚胎口面部区域胶原蛋白代谢的紊乱会导致腭裂的产生。尽管几种类型的胶原蛋白已定位在胚胎腭中，但尚未确定负责调节其合成的因子。转化生长因子β（TGFβ）已被证明能够调节其他组织中的ECM代谢，并且已定位在发育中的腭中。因此，我们在体外研究了TGFβ1调节小鼠胚胎腭间充质（MEPM）细胞中胶原蛋白合成和降解的能力。免疫组织化学分析证实，III型胶原蛋白在胚胎腭间充质中占主导地位，而I型胶原蛋白在整个腭上皮和间充质中普遍存在。通过测量L-[2-3-4-5-³H]脯氨酸掺入羟脯氨酸来测定TGFβ处理的汇合MEPM细胞在无血清条件培养基中的总胶原蛋白产量。用TGFβ1处理24小时可刺激其掺入细胞层和培养基部分。通过ELISA对胶原蛋白类型进行定量分析表明，TGFβ1早在处理后3小时就刺激了III型胶原蛋白的积累。对用TGFβ1处理的MEPM细胞进行Northern印迹分析表明，编码前胶原α1（I）和α1（III）的mRNA的稳态水平升高，并且这些作用被环己酰亚胺消除，但不被放线菌素消除。TGFβ处理对MEPM细胞胶原蛋白水平的影响也反映了胶原蛋白降解的变化。经TGFβ处理的MEPM细胞的总蛋白酶活性显著降低。此外，底物凝胶电泳分析表明脊椎动物胶原酶和基质溶解素特异性降低。这些数据代表了腭发育过程中蛋白水解谱变化的首次报道。因此，TGFβ在合成和降解水平上调节胚胎腭组织中胶原蛋白的含量。
