Interaction of estrogen receptor α with protein kinase C α and c-Src in osteoblasts during differentiation

In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor α (ERα) binding capacity decreases upon confluence. We investigated potential interactions between ERα and PKC isoforms and their confluence-induced modulations in clonal ROS.SMER#14 cells and primary osteoblasts. In sub-confluent ROS.SMER#14 cells, which express an exogenous plus small amounts of the endogenous ERα gene, the receptor appeared as two main bands of ≈66 and ≈46 kDa. In over-confluent, more differentiated cells, the cytosolic ≈66 kDa ERα appeared decreased and the ≈46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCα and PKC , but not PKCζ, was evidenced at over-confluence, along with transient increases in expression and kinase activity of c-Src, accompanied by membrane translocation of the kinase-activated enzyme. In contrast, negligible membrane translocation of PKCα and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERα. PKCα from over-confluent cells phosphorylated p60c-Src in vitro, suggesting functional interaction between the two kinases. ERα co-immunoprecipitated c-Src and PKCα, mostly in its cleaved form (PKMα). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCα/PKMα was ERα-co-immunoprecipitated at over-confluence, a condition in which the shorter, ≈46 kDa ERα variant is increased. This interaction was enhanced by estradiol treatment or PKC down-regulation, but was unaffected by c-Src inhibition. These data highlight direct PKCα–c-Src–ERα interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.