FACTORS AND GENES RESPONSIBLE FOR ADIPOCYTE COMMITMENT

描述：长期目标是确定脂肪组织血管基质的多能干细胞向脂肪细胞谱系承诺的机制。其具体目的是：(1)鉴定BMP4诱导的基因和蛋白质；(2)鉴定BMP4作用于C3H10TI/2多能干细胞后，丝氨酸、苏氨酸或酪氨酸残基发生磷酸化的分子。2.确定上述基因的表达是否导致在细胞培养或体内环境中对脂肪细胞谱系的承诺，并确定这种承诺的机制基础。3.使用蛋白质组学方法鉴定启动或参与承诺过程的分泌蛋白。 BMP4诱导的基因表达的变化将在mRNA转录和蛋白水平上进行检测。在转录水平上，表达的变化将通过寡核苷酸阵列进行监测。由于mRNA和蛋白质水平并不总是反映转录后机制的调节，蛋白质水平的差异也将通过基于质谱学的蛋白质组学方法直接评估。这种结合的方法不仅应该提供转录本数据的验证，而且还可以识别其mRNA水平不变的分子，否则将被基于微阵列的方法遗漏。基于质谱学的方法的其他优点包括识别经过翻译后修饰的蛋白质，例如BMP4诱导的磷酸化，以及通过从头测序识别新的分子。 我们的研究应该得到脂肪细胞承诺过程的详细地图，因为我们不仅应该能够识别影响这一过程的细胞外因素，而且应该能够阐明涉及的细胞内信号通路。

DESCRIPTION: The long-term goal is to determine the mechanisms by which pluripotent stem cells of the vascular stroma of adipose tissue undergo commitment to the adipocyte lineage. The SPECIFIC AIMS are: 1. (A) To identify BMP4-induced genes and proteins, and (B) to identify molecules that undergo phosphorylation on serine, threonine or tyrosine residues upon BMP4 treatment in C3H10TI/2 pluripotent stem cells. 2. To determine whether expression of genes "identified" above causes commitment to the adipocyte lineage in cell culture or in vivo contexts and to determine the mechanistic basis for such commitment. 3. To identify, using a proteomic approach, secreted proteins that initiate, or are involved in, the commitment process. Changes in gene expression induced by BMP4 will be examined at both the mRNA transcript and protein levels. At the transcript level, changes in expression will be monitored with oligonucleotide arrays. Since mRNA and protein levels do not always reflect regulation by post-transcriptional mechanisms, differences in protein levels will also be directly assessed by mass spectrometry-based proteomic methods. This combined approach should not only provide verification of the transcript data, but also allow identification of molecules whose mRNA levels do not change and would otherwise be missed by microarray-based methods. Other advantages of mass spectrometry-based methods include identification of proteins that undergo post-translational modifications such as BMP4-induced phosphorylation and identification of novel molecules by de novo sequencing. Our studies should result in a detailed map of the adipocyte commitment process, as we should not only be able to identify the extracellular factors that influence this process but also elucidate the intracellular signaling pathways that are involved.