Anakysis of GATA-3 function in proliferation and differentiation of kerationocytes

在表皮角质形成细胞中，我们发现GATA-3在分化的角质形成细胞中比未分化的角质形成细胞中更丰富，这表明GATA-3可能参与分化特异性基因调控。我们之前发现c-Fos和Sp1在分化的角质形成细胞中激活loricrin启动子活性，而c-Jun和Sp3在未分化的角质形成细胞中抑制它。因此，我们测试了GATA-3、c-Fos和Sp1对氯丙林启动子活性的协同作用。本研究的反式激活实验证明，即使GATA-3结合基序从启动子中删除，GATA-3、c-Fos和Sp1也能以协同方式激活氯丙氨酸启动子转录。这些结果有力地说明了这些蛋白质之间的相互作用。因此，我们研究了GATA-3、AP-1和Sp1/Sp3之间的物理结合。体外结合谷胱甘肽s -转移酶拉下实验表明，c-Jun/c-Fos可直接与GATA-3结合。然而，GATA-3与Sp1的结合亲和力远低于与c-Fos的结合亲和力，这些结果表明GATA-3可以直接与c-Fos相互作用，而GATA-3可能通过其他转录因子或辅因子与Sp1相互作用。

In epidermal keratinocytes, we demonstrated that GATA-3 was more abundant in differentiated keratinocytes than undifferentiated keratinocytes, suggesting that GATA-3 might contribute to differntiatioirspecific gene regulation. We previously showed that c-Fos and Sp1 activate the loricrin promoter activity in differentiated keratinocytes while c-Jun and Sp3 suppress it in undifferentiated keratinocytes. Thus, we tested the cooperative tarnsactivation of loricrin promoter activity by GATA-3, c-Fos and Sp1. Transactivation experiments presented here provide evidence that GATA-3, c-Fos and Sp1 can activate the loricrin promoter transcription in a synergistic manner even though the GATA-3 binding motif is deleted from the promoter. These results strongly suggest the interaction among those proteins. Thus, we examined the physical binding among GATA-3, AP-1 and Sp1/Sp3. In vitro binding by means of a glutathione S-transferase pull down assay showed that c-Jun/c-Fos could directly associate with GATA-3. However, binding affinity of GATA-3 with Sp1 was much less than that with c-Fos, These results suggest that GATA-3 can interact with c-Fos directly while GATA-3 may interact with Sp1 via other transcription factors or cofactors.