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
Maurizio
M
Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, Italy.
Brama
Marina
M
Marino
Maria
M
Bernardini
Silvia
S
Korach
Kenneth S
KS
Wetsel
William C
WC
Scandurra
Roberto
R
Faraggiana
Tullio
T
Spera
Giovanni
G
Baron
Roland
R
Teti
Anna
A
Migliaccio
Silvia
S
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D015536:Down-Regulation; D047628:Estrogen Receptor alpha; D004967:Estrogens / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*
D047628:Estrogen Receptor alpha; D004967:Estrogens; D011960:Receptors, Estrogen; D019000:Phosphotyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha