S. Migliaccio et al., ENDOGENOUS PROTEIN-KINASE-C ACTIVATION IN OSTEOBLAST-LIKE CELLS MODULATES RESPONSIVENESS TO ESTROGEN AND ESTROGEN-RECEPTOR LEVELS, Molecular endocrinology, 7(9), 1993, pp. 1133-1143
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.