Only the large soluble form of preadipocyte factor-1 (Pref-1), but not the small soluble and membrane forms, inhibits adipocyte differentiation: role of alternative splicing
Only the large soluble form of preadipocyte factor-1 (Pref-1), but not the small soluble and membrane forms, inhibits adipocyte differentiation: role of alternative splicing
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DOI：
10.1042/bj3640137
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发表时间：
2002-05-15
影响因子：
4.1
通讯作者：
Sul, HS
中科院分区：
文献类型：
--
作者：
Mei, BS;Zhao, L;Sul, HS
我们最初将前脂肪细胞因子 1 (Pref-1) 确定为脂肪生成的抑制剂，因为全长 Pref-1A 的组成型表达会抑制 3T3-L1 细胞向脂肪细胞的分化。随后，我们发现 Pref-1 的膜形式在胞外域的两个位点进行蛋白水解加工，产生较大的 (50 kDa) 和较小的 (25 kDa) 可溶形式。尚未阐明具有抑制脂肪细胞分化活性的 Pref-1 的具体形式。在这里，Pref-1的各种人工构建体和选择性剪接变体被稳定转染到3T3-L1细胞中，或者将来自用各种形式转染的COS细胞的条件培养基添加到分化的3T3-L1细胞中。通过油红0染色来判断脂质积累和脂肪细胞标记物的表达，我们确定，与全长Pref-1A和构建的大可溶形式不同，缺少膜近端加工位点的Pref-1人工膜形式不能有效抑制脂肪生成。此外，来自仅含有前三个表皮生长因子重复序列（对应于小可溶形式）的构建体转染的COS细胞的条件培养基不能有效抑制脂肪细胞分化。在四种可变剪接产物中，Pref-1A和Pref-1B同时产生大和小可溶形式，抑制脂肪生成，而Pref-1C和Pref-1D缺乏靠近膜的加工位点，因此仅产生较小的可溶形式，没有显示出任何效果。我们得出的结论是，只有大的可溶形式，而不是跨膜或小可溶形式，Pref-1 具有生物活性，因此选择性剪接决定了 Pref-1 在脂肪细胞分化中的功能。
We originally identified preadipocyte factor-1 (Pref-1) as an inhibitor of adipogenesis by the fact that constitutive expression of full-length Pref-1A inhibits differentiation of 3T3-L1 cells into adipocytes. Subsequently, we found that the membrane form of Pref-1 is proteolytically processed at two sites in the extracellular domain, resulting in the larger (50 kDa) and smaller (25 kDa) soluble forms. A specific form(s) of Pref-1, which is active in inhibiting adipocyte differentiation, has not been elucidated. Here, various artificial constructs and alternative-splicing variants of Pref-1 were stably transfected into 3T3-L1 cells, or conditioned media from COS cells transfected with the various forms were added into differentiating 3T3-L1 cells. Judging by Oil Red 0 staining for lipid accumulation and expression of adipocyte markers, we determined that, unlike the full-length Pref-1A and the constructed large soluble form, the artificial membrane form of Pref-1 lacking the processing site proximal to the membrane was not effective in inhibiting adipogenesis. Furthermore, conditioned media from COS cells transfected with the construct containing only the first three epidermal growth factor repeats, corresponding to the small soluble form, was not effective in inhibiting adipocyte differentiation. Of the four alternative-splicing products, Pref-1A and Pref-1B, which generate both large and small soluble forms, inhibited adipogenesis, whereas Pref-1C and Pref-1D, which lack the processing site proximal to the membrane and therefore generate only the smaller soluble form, did not show any effect. We conclude that only the large soluble form, and not the transmembrane or the small soluble form, of Pref-1 is biologically active and that alternative splicing therefore determines Pref-1 function in adipocyte differentiation.