Modulation of human estrogen receptor α F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells

Abstract

The human estrogen receptor a (ERa) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERa F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C a (PKCa). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in overconfluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, Fpromoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERa resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERa signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERa in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.

G protein-coupled receptor 48 upregulates estrogen receptor α expression via cAMP/PKA signaling in the male reproductive tract

“…In human, ER expression in osteoblast cells was upregulated by PKC/c-Src (Longo et al, 2006), and in breast carcinoma cells by ERF-1 (also known as TFAP2C), a member of the AP2 transcription factor family (McPherson et al, 1997). In this study, our results first demonstrate that ER gene expression could be upregulated by Gpr48 via the cAMP-PKA-Creb pathway.…”

Protein Kinase Cα (PKCα) Regulates Bone Architecture and Osteoblast Activity

“…PKCα and ERα physically interact in osteoblastic cells in a complex involving c-Src (23), suggesting a potential mechanism for cooperation between these proteins within the same signaling cascades. Furthermore, PKC signaling reduces ER signaling in overconfluent osteoblastic cells, which attain a more differentiated state (26, 60), potentially acting as a cellular context-specific “break” on ER signaling that is lost in Prkca −/− mice. Although none of the myriad ERα transgenic mice generated thus far have been reported to form intramedullary bone, it is intriguing that ERα deletion only impairs the osteogenic response to loading in female, not male, mice (15, 61).…”

“…PKCα regulates ERα activity in osteoblast-like cells (23, 26) and inhibits Wnt/β-catenin signaling in cancer cell types (27, 28). Exposure to mechanical strain rapidly activates PKCα in osteoblast-like cells (29), and PKC signaling has been implicated in the regulation of various mechanically responsive genes, including the osteoblast differentiation marker osteocalcin (30–32).…”

Equol promotes rat osteoblast proliferation and differentiation through activating estrogen receptor

“…PKC signaling mediates various cellular functions, such as cell proliferation and differentiation, and the signaling of various osteogenic regulators, such as parathyroid hormone and estrogen (Migliaccio et al, 1998;Capiati et al, 1999;Lampasso et al, 2002;Longo et al, 2006). Inhibition of PKCα activity in osteoblasts results in a marked decrease in mitogenactivated protein kinase activity, which is known to play an important role in the proliferation of cells, leading to a significant decrease in proliferation (Lampasso et al, 2002); on the other hand, activation of PKCα promotes human osteoblast proliferation (Villa et al, 2003;Hsieh et al, 2010;Liang et al, 2012).…”