Greene R M, Lloyd M R, Uberti M, Nugent P, Pisano M M
Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Cell Physiol. 1995 Jun;163(3):431-40. doi: 10.1002/jcp.1041630302.
Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP-dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (RI alpha, RII alpha, and RII beta) and catalytic (C alpha) subunits of PKA was examined. RNA extracted from murine embryonic palatal tissue (days 12-14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed RI alpha and C alpha mRNA were seen on all days of gestation examined. RI alpha transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of RII alpha and RII beta mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of RII beta, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of RII beta was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, RII beta was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in RII alpha mRNA levels but had no effect on steady state levels of RII beta or C alpha mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of RI alpha mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and C alpha subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP.
胚胎腭组织的正常生长和分化取决于细胞内cAMP的调控水平。环磷酸腺苷依赖性蛋白激酶（PKA）介导cAMP的生物学活性。PKA同工酶蛋白谱显示胚胎腭组织在发育过程中存在明显的时间变化模式。为了确定腭发育过程中PKA同工酶谱变化的分子基础，研究了PKA调节亚基（RIα、RIIα和RIIβ）和催化亚基（Cα）mRNA的时空表达。通过Northern印迹分析检测从鼠胚胎腭组织（妊娠第12 - 14天）提取的RNA。在所有检测的妊娠天数中均可见到组成性表达的RIα和Cα mRNA的显著水平。体外腭间充质细胞中RIα转录本的丰度明显低于体内腭组织。RIIα和RIIβ mRNA水平在妊娠第12天最高，这一时期腭组织生长显著。此外，还研究了RIIβ在发育中的胚胎腭中的组织分布模式，此前未被描述过。观察到RIIβ在组织分布上有显著的发育变化。该同工酶在妊娠第12天在腭上皮细胞和间充质细胞中均匀分布，但到妊娠第14天，RIIβ主要定位于腭上皮细胞。用福司可林直接激活鼠胚胎腭间充质（MEPM）细胞中的腺苷酸环化酶导致RIIα mRNA水平升高，但对RIIβ或Cα mRNA的稳态水平没有影响。此外，细胞内cAMP水平的升高导致RIα mRNA转录谱的改变。本研究结果记录了鼠胚胎腭组织中编码各种环磷酸腺苷依赖性蛋白激酶调节亚基和Cα亚基的基因的特定表达模式。此外，我们还证明了该激酶在MEPM细胞中响应细胞内cAMP水平升高的适应性变化。
