Molecular Endocrinology, Vol 7, 1133-1143, Copyright © 1993 by Endocrine Society

ARTICLES

Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels

S Migliaccio, WC Wetsel, WM Fox, TF Washburn and KS Korach
Receptor Biology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with 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 mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells 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 been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O- tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose- dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.

This article has been cited by other articles:

				M. Longo, B. Peruzzi, D. Fortunati, V. De Luca, S. Denger, G. Caselli, S. Migliaccio, and A. Teti Modulation of human estrogen receptor {alpha} F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells J. Mol. Endocrinol., December 1, 2006; 37(3): 489 - 502. [Abstract] [Full Text] [PDF]

				R. Clarke, F. Leonessa, J. N. Welch, and T. C. Skaar Cellular and Molecular Pharmacology of Antiestrogen Action and Resistance Pharmacol. Rev., March 1, 2001; 53(1): 25 - 72. [Abstract] [Full Text] [PDF]

				P. V. N. Bodine, H. A. Harris, and B. S. Komm Suppression of Ligand-Dependent Estrogen Receptor Activity by Bone-Resorbing Cytokines in Human Osteoblasts Endocrinology, June 1, 1999; 140(6): 2439 - 2451. [Abstract] [Full Text]

				A. GARCÍA RATO, J. GARCÍA PEDRERO, M. A. MARTÍNEZ, B. DEL RIO, P. S. LAZO, and S. RAMOS Melatonin blocks the activation of estrogen receptor for DNA binding FASEB J, May 1, 1999; 13(8): 857 - 868. [Abstract] [Full Text]

				S. Migliaccio, T. F. Washburn, S. Fillo, H. Rivera, A. Teti, K. S. Korach, and W. C. Wetsel Modulation of Estrogen Receptor Levels in Mouse Uterus by Protein Kinase C Isoenzymes Endocrinology, November 1, 1998; 139(11): 4598 - 4606. [Abstract] [Full Text] [PDF]

				M. B. Martin, P. Garcia-Morales, A. Stoica, H. B. Solomon, M. Pierce, D. Katz, S. Zhang, M. Danielsen, and M. Saceda Effects of 12-O-Tetradecanoylphorbol-13-acetate on Estrogen Receptor Activity in MCF-7 Cells J. Biol. Chem., October 20, 1995; 270(42): 25244 - 25251. [Abstract] [Full Text] [PDF]