Illumina-microarray analysis of mycophenolic acid-induced cell death in an insulin-producing cell line and primary rat islet cells: New insights into apoptotic pathways involved

Abstract

Mycophenolic acid (MPA), widely used to prevent organ transplant rejection, may induce toxicity and impair function in β-cells. Mechanisms of MPA-induced cell death have not been fully explored. In this study, we examined gene expression patterns in INS-1E cells and isolated primary rat islets following MPA treatment using the Illumina-cDNA microarray. The MPA treatment decreases RhoGDI-α gene expression, which points to apoptosis by JNK activation through a MAPKs-dependent pathway. A strong association between RhoGDI-α and Rac1 activation during MPA-induced apoptosis is also consistent with apoptosis through JNK. Suppression of RhoGDI-α using siRNA and gene over-expression both affected the cell death rate, consistent with Rac1 activation and downstream activation of MAPKs signaling. We confirmed that Rac1 protein mediates the interaction between RhoGDI-α and JNK signaling. We conclude that MPA-induced cell death in primary β-cells and an insulin-secreting cell line proceeds through RhoGDI-α down-regulation linked to Rac1 activation, with subsequent activation of JNK. The RhoGDI-α/Rac1/JNK pathway may present a key to intervention in MPA-induced islet apoptosis.

Introduction

In type 1 diabetes mellitus, an autoimmune response selectively destroys insulin-secreting β-cells in the islets of Langerhans, with potentially fatal insulin depletion [1], [2]. Trials show that islet cell transplantation may cure type 1 diabetes. Although clinically more reliable and reproducible than pancreas transplantation, islet transplantation presents several problems. In particular, the transplants are susceptible to drug-induced toxicity and death, and may prove physiologically inadequate [3]. Strategies to overcome these obstacles include development of less toxic immunosuppressant drugs and agents to modulate the toxic drug response. [4].

Mycophenolic acid (MPA), a widely used immunosuppressant, inhibits inosine 5′-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo guanosine nucleotide synthesis [5], [6]. In pancreatic organ or islet transplants, MPA inhibits β-cell function and induces cell death through guanosine triphosphate depletion [7], [8].

Rho proteins act as signaling components in cell proliferation, adhesion, motility, differentiation and apoptosis [9]. Rho proteins mediate critical steps in MPA-induced cell death, but the mechanisms for this are not clear. In mammals, the Rho family contains about 20 subfamilies, but most functional data concern Rac, Rho, and Cdc42 only [10]. Rac and Cdc42 may be dysregulated in carcinogenesis and metastasis, as well as in apoptotic cell death, in many different cell types [11]. Most Rho/Rac GTPases behave as “molecular switches” that shift between inactive and active conformations through hydrolysis of GTP [12], [13]. Three types of proteins regulate the RhoGTPase activities: (i) guanine nucleotide exchange factors (GEFs) stimulate the GTP–GDP exchange reaction; (ii) GTPase-activating proteins (GAPs) mediate GTP hydrolysis; and (iii) guanine nucleotide dissociation inhibitors (GDIs) bind to Rho GTPases and block the dissociation of GDP from the GTP-binding site [14]. In addition, three types of GDI, including RhoGDI-α [15], [16], RhoGDI-β (or Ly/D4GDI) [17], and RhoGDI-γ [18], regulate RhoGTPase in specific cell types. Tumor cells of ovarian, breast and hepatic origin usually over-express RhoGDI-α, and mechanisms of apoptosis induced by the etoposides and doxorubicin may involve RhoGDI-α [19], [20]. RhoGDI-α may form one-to-one complexes with isoprenylated RhoA, RhoB, Rac1, Rac2, Cdc42, and express distinct functions in each association. Rac1, for example, mediates apoptosis in intestinal epithelial cells as a signaling component in the JNK pathway [16], [21], [22], [23]. By inhibiting dissociation of Rac1 and GDP, RhoGDI-α participates in the apoptotic pathways of several cell types [21], [22]. RhoGDI-α may also inhibit cleavage of Rac1 by caspases, which is required for the maximal apoptotic response to cytotoxic drugs.

Previously, we showed that MPA induces apoptosis in HIT-T15 by increasing JNK activity in a time- and dose-dependent manner [23]. We also found that MPA induces significant apoptosis in an insulin-secreting cell line, RIN-5F, through RhoGDI-α down-regulation linked with an increase in JNK expression [24].

Based on these findings, we hypothesized that genes differentially expressed during MPA-induced apoptosis may correspond to specific cellular events in insulin-secreting cells, and that in vivo and in vitro assays based on these data may be used to test the level of function in islet transplants. We focused initially on RhoGDI-α, which is differentially expressed after MPA treatment. We also observed that RhoGDI-α over-expression prevented MPA-induced cell death and decreased levels of activated JNK and MKK4/7, whereas RhoGDI-α knock-down enhanced MPA-induced cell death through MAPK activation. In this study, we found that RhoGDI-α directly affected MPA-induced apoptosis in INS-1E cells and in primary rat islet cultures via Rac1/MAPK/JNK signaling. Rac1 thus emerged as a critical intermediary between RhoGDI-α and the MAPK signaling pathway.