Benzopyrene activates SAPK and induces HAND1 that favors differentiation of trophoblast stem cells

Abstract

OBJECTIVE: To test whether cigarette tar BaP causes mouse trophoblast stem cells (TSC) to activate stress-activated protein kinase/jun kinase (SAPK/JNK) and the transcription factor Heart and Mesoderm inducer (HAND)1.DESIGN: Culture TSC with or without BaP, fractionate proteins for size using SDS-PAGE, use antibodies to phosphorylated SAPK Thr183/Tyr185 or HAND1 to validate antibodies and test for regulation, then use antibodies by epifluorescence methods to study TSC subpopulations expressing HAND1.MATERIALS AND METHODS: We used immunoblot to analyze phosphorylated SAPK Thr183/Tyr185 and HAND1 expression in TSC using polyclonal rabbit antibody. ICC was used to determine the dose and time-dependent effects of stress using sorbitol on phosphorylated SAPK Thr183/Tyr185 and HAND1 in TSC nuclei. The effect of stress induced for 1hr and 24 hrs with and without BaP was analyzed with SAPK Thr183/Tyr185 and HAND1 antibodiesRESULTS: Phosphorylated SAPK and HAND1 proteins at 46/54kDa and 30kDa, respectively, were induced by BaP in a time- and dose-dependent manner in cultured TSC. The threshold BaP dose for HAND1 induction and decreased cell accumulation was the same.CONCLUSIONS: Benzo(a)pyrene from cigarette smoke, is stressful in activating SAPK in TSC and also can lead to increased HAND1 protein that is known to be necessary for induction of placental lactogen-1, the first placental hormone produced by the implanting mouse embryo. Interestingly, the threshold dose for inducing HAND1 is the same as that previously determined to induce significant ID2 loss and decrease in TSC accumulation. Thus, BaP predisposes mouse TSC to differentiate to compensate for insufficient TSC accumulation, suggesting that cigarette smoke can cause TSC differentiation. Since SAPK is activated in embryos and TSC by many stressors, it is likely that an improper balance in TSC potency/differentiation is part of a novel but widespread etiology for placental insufficiency and disease. OBJECTIVE: To test whether cigarette tar BaP causes mouse trophoblast stem cells (TSC) to activate stress-activated protein kinase/jun kinase (SAPK/JNK) and the transcription factor Heart and Mesoderm inducer (HAND)1. DESIGN: Culture TSC with or without BaP, fractionate proteins for size using SDS-PAGE, use antibodies to phosphorylated SAPK Thr183/Tyr185 or HAND1 to validate antibodies and test for regulation, then use antibodies by epifluorescence methods to study TSC subpopulations expressing HAND1. MATERIALS AND METHODS: We used immunoblot to analyze phosphorylated SAPK Thr183/Tyr185 and HAND1 expression in TSC using polyclonal rabbit antibody. ICC was used to determine the dose and time-dependent effects of stress using sorbitol on phosphorylated SAPK Thr183/Tyr185 and HAND1 in TSC nuclei. The effect of stress induced for 1hr and 24 hrs with and without BaP was analyzed with SAPK Thr183/Tyr185 and HAND1 antibodies RESULTS: Phosphorylated SAPK and HAND1 proteins at 46/54kDa and 30kDa, respectively, were induced by BaP in a time- and dose-dependent manner in cultured TSC. The threshold BaP dose for HAND1 induction and decreased cell accumulation was the same. CONCLUSIONS: Benzo(a)pyrene from cigarette smoke, is stressful in activating SAPK in TSC and also can lead to increased HAND1 protein that is known to be necessary for induction of placental lactogen-1, the first placental hormone produced by the implanting mouse embryo. Interestingly, the threshold dose for inducing HAND1 is the same as that previously determined to induce significant ID2 loss and decrease in TSC accumulation. Thus, BaP predisposes mouse TSC to differentiate to compensate for insufficient TSC accumulation, suggesting that cigarette smoke can cause TSC differentiation. Since SAPK is activated in embryos and TSC by many stressors, it is likely that an improper balance in TSC potency/differentiation is part of a novel but widespread etiology for placental insufficiency and disease.