Mono-(2-ethylhexyl) phthalate rapidly increases celsr2 protein phosphorylation in HeLa cells via protein kinase C and casein kinase 1.

Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Some features of phthalate-induced testicular injury suggest that phthalates alter Sertoli-germ cell adhesion and G protein signaling. Celsr2 is a unique protein that has structural characteristics of both an adhesion molecule and a G protein coupled receptor (GPCR) and has been demonstrated to function in Sertoli-germ cell adhesion. Within 2 h of a 1-g/kg mono-(2-ethylhexyl) phthalate (MEHP) exposure, in vivo Sertoli cell celsr2 localization was altered; celsr2 immunostaining became concentrated in the basal aspect of Sertoli cells, and then a diffuse pattern emerged. Because GPCRs are regulated by phosphorylation, the hypothesis that phthalate exposure induces the phosphorylation of celsr2 was tested by examining phosphorylation in celsr2-transfected HeLa cells treated with MEHP. At concentrations of 1 microM or greater, MEHP transiently increased celsr2 phosphorylation on serine/threonine residues; celsr2 phosphorylation was increased by 15 min of exposure and returned to control levels after 60 min. Cells exposed to the inactive phthalate monoester mono-methyl phthalate showed no change in celsr2 phosphorylation. In addition, phosphorylation of the endogenous HeLa cell GPCR, Chemokine Receptor 4 (CXCR4), was not altered by exposure to MEHP. Inhibition of protein kinase C or casein kinase 1 prevented MEHP-induced celsr2 phosphorylation, while inhibition of protein kinase A or mitogen-activated protein kinase had no effect. These data show that MEHP exposure rapidly alters testicular celsr2 immunolocalization as well as celsr2 posttranslational modification in a model cell line.