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.
Division of Biological Sciences, CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709, USA.
Stephanie A
SA
Lahousse
Stephanie A
SA
Beall
Kamin J
KJ
Johnson
D015820:Cadherins / Q000378:metabolism*; D047389:Casein Kinase I / Q000378:metabolism*; D004051:Diethylhexyl Phthalate / Q000031:analogs & derivatives* / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism
C498477:CELSR2 protein, human; D015820:Cadherins; D019718:Receptors, CXCR4; D004051:Diethylhexyl Phthalate; D047389:Casein Kinase I; D011493:Protein Kinase C; C016599:mono-(2-ethylhexyl)phthalate