Transcriptional regulation of leptin in adipocytes.

我们在大鼠成熟脂肪细胞和人滋养细胞BeWo细胞的原代培养中检测了人瘦素5'-侧翼序列的启动子活性。将不同长度的人瘦素5′侧翼序列亚克隆到无启动子萤火虫荧光素酶表达载体中。在大鼠成熟脂肪细胞中，最长的启动子（-2080到+ 108）结构显示出高水平的转录活性。当从-2080到-136进行删除时，没有注意到启动子活性的显著变化。这个136 bp的区域包含3个Sp1结合位点和一个C/EBP结合位点。当Sp1位点和C/EBP位点同时突变时，启动子活性降低到无启动子结构的水平。在BeWo细胞中，最长的启动子（-2080到+108）结构也显示出高水平的转录活性。从-2080到-1885的删除没有显著的变化。然而，当-1885和-1830之间的序列被删除时，启动子活性下降了80%以上。电泳迁移位移分析显示，在BeWo细胞中存在与-1885至-1830序列结合的核蛋白，而在大鼠成熟脂肪细胞中不存在。我们还研究了蛋白激酶A和C对脂肪细胞和BeWo细胞瘦素产生的影响。福斯克林减少瘦素的产生，而PMA没有改变。福斯克林增加了BeWo细胞的瘦素产量。蛋白激酶a的抑制剂H89完全抑制福斯克林诱导的瘦素生成的增加，肉豆蔻酸酯（PMA）增加BeWo细胞的瘦素生成。pma诱导的瘦素产生的增加被H7和staurosporine完全抑制，两者都是蛋白激酶c的抑制剂。本研究表明，人瘦素基因转录在脂肪细胞和胎盘滋养细胞之间受到不同的调节。

We examined the promoter activity of the human leptin 5'-flanking sequences in primary cultures of rat mature adipocytes and BeWo cells, a human trophoblast cell line. Various length of the human leptin 5'-flanking sequences were subcloned into the promoterless firefly luciferase expression vector. In rat mature adipocytes, the longest promoter (-2080 to + 108) construct showed a high-level transcription activity. When deletion proceeded from -2080 to -136, no significant changes in promoter activity were noted. This 136-bp region contained three Sp1 binding sites and a C/EBP binding site. When the Sp1 sites and C/EBP site were simultaneously mutated, the promoter activity was reduced to that of the promoterless construct. In BeWo cells, the longest promoter (-2080 to +108) construct also showed a high-level transcription activity. Deletion from -2080 to -1885 made no significant changes. However, the promoter activity was decreased by more than 80%, when sequences between -1885 and -1830 were deleted. Electrophoretic mobility shift assays revealed the presence of nuclear protein(s) binding to the sequences (-1885 to -1830) in BeWo cells but not in rat mature adipocytes. We also investigated the effect of protein kinases A and C on leptin production in adipocytes and BeWo cells. Leptin production was decreased by forskolin but unchanged by PMA.Leptin production in BeWo cells was increased by forskolin. The forskolin-induced increase in leptin production was completely suppressed by H89, an inhibitor of protein kinase A.Leptin production in BeWo cells was increased by phorbol myristate acetate (PMA). The PMA-induced increase in leptin production was completely suppressed by H7 and staurosporine, both of which are inhibitors of protein kinase C.The present study demonstrates that the human leptin gene transcription is differentially regulated between adipocytes and placental trophoblasts.