The Recombination Of Different Domains Of Gst-smad4Fusion Protein And Their Interaction With Estrogen Receptor β

Prostate cancer is one of the most common cancers in men in the world, and its initiation and progression are relevant to TGF-(3and steroid hormone signal pathways. TGFβ-Smad signal pathway is an important apoptotic pathway in normal prostate tissue and is inactivated in prostate cancer, especially in hormone-independent prostate cancer. Steroid receptors, especially androgen receptor (AR) and estrogen receptor (ER) play important roles in the development and progression of prostate cancer; but the situation that androgen receptor (AR) lost in hormone-independent prostate cancer, and ERβ (estrogen receptor beta) commonly expressed in prostate cancer indicated the potential important functions of ERP in prostate cancer.The aim of the present study is to identify potential crosstalk between the signal Smad signal pathway and ERP signal pathway. As Smad4is the central mediator in TGFβ-Smad signaling, we studied the interaction between different domains of Smad4and full length ERp.To pursue this objective, Smad4was divided into three different domains that were separately named MH1domain, Linker region and MH2domain. These three domains of human Smad4were over-expressed in the Glutathione S-transferase Gene Fusion System. Based on the reasonable amounts of soluble Smad4domains, we explored the details of the interactions between Smad4and endogenous ERβ in prostate cancer DU145cells. Firstly, we constructed pGEX-4T-1/hSmad4MH1domain (DNA sequence from1to438bp), pGEX-4T-1/hSmad4linker domain (DNA sequence from439to924bp), and pGEX-4T-1/hSmad4MH2domain (DNA sequence from925to1659bp) recombinant plasmids and transformed them into Escherichia coli BL21cells. Then the GST-Smad4MH1domain, GST-Smad4linker region and GST-Smad4MH2domain fusion proteins were induced by IPTG and further purified by Glutathione Sepharose4B to obtain the right size fusion proteins. Finally, ERP was extracted from prostate cancer DU145cell lysates and the interaction between Smad4and ERβ was assessed by GST pull-down. Our result showed that the MH2domain of Smad4but not other domains of Smad4could interact with estrogen receptor β. So, our study indicate the crosstalk between the TGF-β/Smad signal and ERβ signal pathways, and provide the basis for further research on the functions of these two important pathways in prostate cancer development.