Constance C M, Morgan J I, Umek R M
Department of Biology, University of Virginia, Charlottesville, 22903, USA.
Mol Cell Biol. 1996 Jul;16(7):3878-83. doi: 10.1128/MCB.16.7.3878.
CCAAT/enhancer-binding protein alpha (C/EBPalpha) is expressed in postmitotic, differentiated adipocytes and is required for adipose conversion of 3T3-L1 cells in culture. Temporal misexpression of C/EBPalpha in undifferentiated adipoblasts leads to mitotic growth arrest. We report here that growth arrest- and DNA damage-inducible gene 45 (gadd45) is preferentially expressed in differentiated 3T3-L1 adipocytes similar to phenotype-associated genes. Furthermore, C/EBPalpha transactivates a reporter plasmid containing 1.5 kb of the gadd45 promoter region. The proto-oncogene myc, which inhibits adipocyte differentiation, abrogates C/EBPalpha activation of gadd45. gadd45 is known to be a target of the tumor suppressor p53 in a G1 checkpoint activated by DNA damage. Immunoprecipitation of radiolabeled proteins with conformation-specific antibodies revealed that wild-type p53 is expressed throughout 3T3-L1 adipocyte development, including the postmitotic period characterized by the accumulation of gadd45 and C/EBPalpha. A stable 3T3-L1 subline was engineered to express a dominant negative p53, human p53(143ala). The p53(143ala) subline differentiated to adipocytes and showed appropriate developmental expression of gadd45. These findings suggest that postmitotic growth arrest is coupled to adipocyte differentiation via C/EBPalpha stimulation of growth arrest-associated and phenotype-associated genes.
CCAAT/增强子结合蛋白α（C/EBPα）在有丝分裂后分化的脂肪细胞中表达，是培养的3T3-L1细胞脂肪转化所必需的。C/EBPα在未分化的脂肪母细胞中的短暂错误表达会导致有丝分裂生长停滞。我们在此报告，生长停滞和DNA损伤诱导基因45（gadd45）在分化的3T3-L1脂肪细胞中优先表达，类似于与表型相关的基因。此外，C/EBPα可反式激活包含1.5 kb gadd45启动子区域的报告质粒。抑制脂肪细胞分化的原癌基因myc可消除C/EBPα对gadd45的激活作用。已知gadd45是DNA损伤激活的G1检查点中肿瘤抑制因子p53的靶点。用构象特异性抗体对放射性标记蛋白进行免疫沉淀显示，野生型p53在3T3-L1脂肪细胞发育的整个过程中都有表达，包括以gadd45和C/EBPα积累为特征的有丝分裂后时期。构建了一个稳定的3T3-L1亚系以表达显性负性p53，即人p53（143ala）。p53（143ala）亚系分化为脂肪细胞，并显示出gadd45的适当发育表达。这些发现表明，有丝分裂后生长停滞通过C/EBPα对生长停滞相关基因和表型相关基因的刺激与脂肪细胞分化相关联。
