the role of adaptor protein SH2-B in adipocyte differentiation

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Expression of SH2-B splicing variants in 3T3-L1 cells during adipocyte differentiation.Preadipocyte cell line mouse 3T3-L1 is extensively used to investigate the molecular mechanisms in adipogenesis. To evaluate adipocyte differentiation, three different methods such as Oil red-O staining, RT-PCR for adipocyte specific genes and GPDH assay, were used. Among 4 isoforms of SH2-B, expression of β- andγ- isoforms were detected in 3T3-L1 cells, and these expression levels were up-regulated during adipogenesis. Expression of SH2-B family proteins was also investigated. Changes in expression level of APS was similar to these of SH2-B splicing variants, although mRNA level of lnk did not change during adipogenesis.Effect of overexpression or suppression of SH2-B in 3T3-L1 cells on adipocyte differentiation.Willd-type and mutated human SH2-Bβ were introduced into 3T3-L1 cells by using a lipofection method. The expression of human SH2-B was confirmed by RT-PCR and western blot analysis. The expression of wild-type SH2-B accelerated the cellular differentiation into adipocytes. On the other hand, the mutated SH2-B, which lacks the ability to bind to phosphorylated proteins, suppressed its differentiation. The expression of small interfering RNA against SH2-B also inhibited adipogenesis in 3T3-L1 cells.In conclusion, these results clearly indicate that SH2-B plays a pivotal role in adipocyte differentiation.