Effects of overexpression of CXCL10 (cytokine-responsive gene-2) on MA-10 mouse Leydig tumor cell steroidogenesis and proliferation

Abstract

Chemokines have been implicated in tumor growth, angiogenesis, metastasis and the host immune response to malignant cells. Infection and autoimmune disorders can reduce androgen production by Leydig cells and adversely affect spermatogenesis. Cytokine-responsive gene-2 (crg-2) (systematic name CXCL10, also known as interferon-gamma-inducible protein 10 (IP-10)) is a potent chemokine expressed predominantly by macrophages and Leydig cells in the testis. CXCL10 binds to CXCR3 receptor (a G-protein-coupled receptor) and acts via Gialpha protein. We have shown previously that CXCL10 is differentially expressed in normal Leydig cells, inhibited by human chorionic gonadotropin and induced by interferon-gamma, interleukin-1alpha and tumor necrosis factor-alpha. The purpose of the present study was to determine the effects of overexpression of CXCL10 by transfection experiments in MA-10 cells on cell growth, CXCR3 expression, progesterone synthesis and steroidogenic acute regulatory protein (StAR D1, a key regulatory factor in steroidogenesis) gene expression. We cloned the complete CXCL10 cDNA in a mammalian expression vector with the CMV promoter, pcDNA3.1D/V5-His-TOPO, and confirmed its expression with rat CXCL10 antibody and V5 antibody. Results showed large amounts of CXCL10 protein secreted in the medium in the CXCL10 transfectants by Western blotting. The production of CXCL10 mRNA ranged from 30-50-fold more (n=6) in the transfected cells than the control cells, as determined by semiquantitative and real-time RT-PCR. 8-Br-cAMP downregulated CXCL10 mRNA expression and stimulated CXCR3 mRNA expression. Transfection of MA-10 cells with CXCL10 decreased cAMP-induced progesterone synthesis from 38.5+/-1.7 ng/ml (1.5 x 10(5) cells/ml) in control cells to 23.2+/-1.5 ng in transfected cells (P<0.01). 8-Br-cAMP (0.2 mM)-induced StAR D1 mRNA was decreased 30-40% by transfection with CXCL10. Interestingly, overexpression of CXCL10 induced the expression of its receptor CXCR3 gene, as determined by RT-PCR and fluorescence-activated cell sorter (FACS) analysis. Transfection of CXCL10 also significantly decreased insulin-like growth factor-I (IGF-I, 100 ng/ ml)-induced [3H]thymidine incorporation into DNA. These data suggest that CXCL10 also inhibits MA-10 tumor cell proliferation. In conclusion, CXCL10 inhibits StAR D1 expression, decreases progesterone synthesis and inhibits cell proliferation. CXCL10 has the potential to be used in gene therapy for prostate cancer due to its antiangiogenic effect and its inhibitory effect on steroidogenesis.