Interaction of estrogen receptor alpha with protein kinase C alpha and c-Src in osteoblasts during differentiation.
In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor alpha (ERalpha) binding capacity decreases upon confluence. We investigated potential interactions between ERalpha 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 ERalpha gene, the receptor appeared as two main bands of approximately 66 and approximately 46 kDa. In over-confluent, more differentiated cells, the cytosolic approximately 66 kDa ERalpha appeared decreased and the approximately 46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCalpha and PKCepsilon, but not PKCzeta, 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 PKCalpha and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERalpha. PKCalpha from over-confluent cells phosphorylated p60c-Src in vitro, suggesting functional interaction between the two kinases. ERalpha co-immunoprecipitated c-Src and PKCalpha, mostly in its cleaved form (PKMalpha). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCalpha/PKMalpha was ERalpha-co-immunoprecipitated at over-confluence, a condition in which the shorter, approximately 46 kDa ERalpha 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 PKCalpha-c-Src-ERalpha interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.
Longo, M; Brama, M; Marino, M; Bernardini, S; Korach, KS; Wetsel, WC; Scandurra, R; Faraggiana, T; Spera, G; Baron, R; Teti, A; Migliaccio, S
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