ENDOGENOUS PROTEIN-KINASE-C ACTIVATION IN OSTEOBLAST-LIKE CELLS MODULATES RESPONSIVENESS TO ESTROGEN AND ESTROGEN-RECEPTOR LEVELS

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected wit h the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence , but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanis ms underlying this loss of responsiveness in these ER stably transfect ed cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the sub confluent cells were actively dividing, whereas none of the postconflu ent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cell s was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has bee n reported to influence cell proliferation and steroid hormone recepto r binding, PKC activity was measured in sub- and post-confluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no difference s were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were tr eated with PKC inhibitors (H-7 or staurosporine) or with the tumor pro moter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulat ion of the PKC activity in postconfluent cells enhanced ER-binding cap acity in a dose-dependent manner and estrogen responsiveness of an exo genous reporter gene and of the endogenous alkaline phosphatase, repre senting an endogenous estrogen-stimulated gene. These data indicate th at there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the prolife rative and/or differentiative state of the cells, resulting in modulat ion of hormone responsiveness.