The type 1 diabetes candidate gene CTSH protects against cytokine-induced β-cell apoptosis via Rac2 GTPase

Background and aims: Genome-wide association studies have identified more than 50 risk loci for type 1 diabetes (T1D), including chr15q25.1 with the candidate gene CTSH (lysosomal protease cathepsin H). We previously showed that T1D-associated risk variants in CTSH affect the expression of CTSH, and that CTSH regulates β-cell function, apoptosis, and disease progression in children with newly-diagnosed T1D. By global gene expression analysis, the aim of this study was to identify the genes and mechanisms through which CTSH mediates its protective effect. Materials and methods: INS-1 cells (n=3) stably transfected with a plasmid encoding CTSH (pCTSH) or an empty vector (pcDNA) were stimulated with IL-1β and IFN-γ for 0, 6 and 16 hours. Microarray analysis was performed on the extracted RNA using Affymetrix Rat Genome 230 array. Data were analyzed using the Bioconductor affy package and limma in R. Pathway analysis was performed using ROntoTools in R which takes into account the log fold changes and p-values for the differentially expressed genes as well as pathways topology in order to assess the abnormal perturbation of the pathways in the condition under study. Two pathways were validated functionally by siRNA-mediated knockdown of Gna15, Cav1 and Rac2 in the pcDNA and pCTSH cells (n=5) to examine the effect on CTSH-mediated protection against apoptosis using the Caspase-Glo 3/7, the CytoTox-Fluor, and the Cell Death Detection ELISA assays. Knockdown was verified by real-time qPCR. Results: A total of 56 annotated genes were differentially expressed between the pCTSH and pcDNA cells (abs fold change>1.2 and FDR-adjusted p<0.05). Six pathways were significantly perturbed by CTSH overexpression (p<0.05): “Serotonergic synapse” with the differentially expressed gene Ptgs1, “Calcium signaling” with Gna15, “Amoebiasis” with C9 and Gna15, “Chagas disease” with Ccl5 and Gna15, “Focal adhesion” with Cav1 and Rac2, and “Insulin resistance” with Ppargc1a, Ppp1r3c and Trib3. The three genes from the “Calcium signaling” and “Focal adhesion” pathways (Gna15, Cav1 and Rac2) were all upregulated in response to CTSH overexpression (p<0.05) and therefore knockdown experiments were done individually to evaluate their potential as mediators of the protective effect of CTSH against apoptosis. Knockdown of Rac2 reversed the protective effects of CTSH overexpression on cytokine-induced apoptosis (p<0.05), whereas knockdown of Gna15 and Cav1 had no effect. Conclusion: The current data suggest that CTSH may mediate its protective effect on cytokine-induced apoptosis through the Rho family small GTPase Rac2, which itself is a T1D candidate gene located at the chr22q12.3 risk locus. Interestingly, Rac1, which has 92% sequence homology with Rac2, is also expressed in insulin-secreting cells, where it has been implicated in both glucose-stimulated insulin secretion and apoptosis. Further experiments are needed to evaluate if higher CTSH and Rac2 levels in β-cells protect against immune-mediated damage and preserve β-cell function, thereby representing possible targets for β-cell therapy in T1D.