RhoGDI-a mediates the distinct cycling of Rac1 to regulate a-cell death after treatment of mycophenolic acid

Yuri Cho³, Ji Hey Do³, Yun-Jong Park³, Kyu Ha Huh^2,3, Myoung Soo Kim^2,3, Dong Jin Joo^2,3, Yu Seun Kim^1,2,3.

¹BK21 for Medical Science, Yonsei University College of Medicine; ²Department of Transplantation Surgery, Severance Hospital; ³The Research Institute for Transplantation, Yonsei University College of Medicine; Seoul, South Korea.

Introduction: Mycophenolic acid (MPA) is an immunosuppressive agent which has been widely used for organ transplantation including the pancreas and islet cells. However, potential toxicity on islets has been observed from the impediment of insulin secretion and reduction of the DNA content in rat islets. Previously, we showed that MPA significantly induces apoptosis in insulin-secreting cells through RhoGDI-α down-regulation linked with an increase in JNK expression. In this study, we have chosen a small GTPase, Rac1, and investigated whether its expression pattern is closely related to RhoGDI-α on MPA-induced apoptosis in the insulin-secreting cell line (INS-1E).

Methods: INS-1E cells were treated with MPA for 12, 24 and 36hrs. Microarray was performed according to the Macrogen rat BeadChip technical manual using Illumina RatRaf-12 Expression BeadChip. Functional screening was checked by using small interference RNA (siRNA)-mediated knockdown and over-expression of RhoGDI-α gene in INS-1E cell line. Immune-precipitation was examined to test the physical interaction between RhoGDI-α and Rac1.

Results: It was found that thousands of genes were altered during MPA-induced apoptosis, and RhoGDI-α gene expression pattern was significantly decreased. RhoGDI-α over-expression significantly suppressed Rac1 expression, and Rac1 silencing with siRNA reduced MPA-induced cell death and altered expression of MKK4/7, p-JNK and cleaved caspase-3. Furthermore, using the Rac1 activation assay and immune-precipitation, we have found that Rac1 and RhoGDI-α expressions are closely correlated.

Conclusion: MPA induces down-expression of RhoGDI-α, which is involved with a small GTPase, Rac1. And then, down-stream pathway of Rac1 activates JNK signaling pathway. This novel pathway may be an important key to explain the mechanism of MPA-related cellular apoptosis in transplanted islet cells.