PPARγ1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion
PPARγ1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion
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DOI：
10.1002/jcb.10743
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发表时间：
2004-02-01
影响因子：
4
通讯作者：
Jefcoate, CR
中科院分区：
文献类型：
--
作者：
Cho, YC;Jefcoate, CR
胰岛素(I)、地塞米松(D)和甲基异丁基黄嘌呤(M)的标准激素组合，结合新的血清更新，典型地刺激小鼠胚胎成纤维细胞(MEF)的脂肪生成。在C3H10T1/2(10T1/2)细胞中，在IDM刺激36h后，早期关键的成脂调节因子--过氧化物酶体增殖物激活受体Gamma1(PPARGamma1)的表达最佳。尽管先前的研究证明3T3-L1细胞的有丝分裂克隆扩增对脂肪细胞的分化是必不可少的，但我们在此表明，10T1/2细胞不需要有丝分裂克隆扩增，而是依赖细胞周期通过S期的进展来致力于脂肪细胞的分化。从标准的IDM方案中排除两个主要的有丝分裂刺激(不含胰岛素的DM和新鲜血清更新)可消除有丝分裂的克隆性扩张，但保持等同的PPARGamma1合成和脂肪生成。在激素刺激下，不同的S期抑制物(阿昔双林、羟基脲、L-含羞草和罗斯科维丁)分别将细胞阻滞在S期，并完全阻断PPAR-Gamma1的合成和脂肪生成。然而，G2/M抑制剂影响IDM刺激细胞的G2/M积聚，并阻止有丝分裂，但完全维持PPARGamma1的合成和脂肪生成。无论有无新鲜血清更新，DM刺激都会增加一部分细胞的DNA合成(通过BrdU标记来测量)，并在没有完全有丝分裂的情况下积累G2/M期细胞周期进程。相比之下，更新新鲜血清的标准IDM治疗可促进DNA合成和有丝分裂克隆扩张，同时达到同等水平的成脂作用。即使在克隆分离的情况下，10T1/2混合细胞群体中至多有一半分化为成熟脂肪细胞。PPARGamma仅在含有脂滴的细胞中表达。在低细胞密度(LD)培养条件下，IDM可促进PPARGamma1的合成和脂肪生成，但对约一半的细胞有刺激作用，且对G1/S敏感，但对G2/M抑制剂无明显作用。重要的是，在所有分化的细胞中都发生了生长停滞，而在未分化的细胞中发生了持续的有丝分裂克隆扩张。无论融合程度如何，10T1/2细胞在进展后通过S期进行分化，在此阶段，IDM刺激诱导的成脂承诺是PPARγ合成和随后脂肪细胞分化的先决条件。
Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I),dexamethasone(D), and methylisobutylxanthine(M), administered with afresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that Mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, L-Mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.