The AP-1 family comprises a group of inducible proteins that bind to a common test or 1-way ANOVA, as appropriate

The AP-1 family comprises a group of inducible proteins that bind to a common test or 1-way ANOVA, as appropriate. stimulating osteoclast formation (1), a process enhanced by several inflammatory cytokines. Among them is TNF- (2, 3), a protein produced mainly by cells of the monocyte/macrophage lineage (4). Once released in the bone microenvironment, TNF stimulates osteoclast formation, in part by inducing the production of M-CSF by bone marrow stromal cells (5, 6). M-CSF, along with osteoprotegerin ligand (OPGL; also known as ODF/TRANCE/RANKL) (7), is an essential stimulator of osteoclast precursor proliferation and differentiation (8C10). Studies in ovariectomized mice, an established experimental model of postmenopausal osteoporosis (1), have provided R788 (Fostamatinib) compelling evidence that increased production of TNF plays a critical causal role in ovariectomy-induced bone loss. This evidence includes the finding that treatment with the TNF inhibitor TNF-binding protein completely prevents ovariectomy-induced bone loss (11) and the report that transgenic mice insensitive to TNF owing to the overexpression of a soluble TNF receptor are also protected against ovariectomy-induced bone loss (12). Evidence has also accumulated that demonstrates that E2 downregulates the production and/or the release of TNF by several cell lineages, including bone marrow monocytes and bone cells. Thus, in monocytes and osteoblasts, TNF protein and TNF mRNA levels are increased after natural or surgical menopause and decrease with E2 replacement (13C15). E2 inhibits TNF production by direct effects on target cells, as demonstrated by the fact that in vitro E2 treatment decreases TNF levels in cultures of murine bone marrow monocytes (16) and peripheral blood monocytes (17) and decreases TNF mRNA R788 (Fostamatinib) expression in a murine monocytic cell line (18). Taken R788 (Fostamatinib) together, these studies demonstrate that the bone-sparing effect of E2 is, at least in part, a result of its ability to downregulate the bone marrow levels of TNF. Although the role of TNF as key enhancer of bone resorption in E2-deficient rodents and humans has been defined, the mechanism by which E2 downregulates TNF production remains to be determined. The TNF gene is transcriptionally silent in unstimulated monocytes and is rapidly transcribed in response to a variety of signals, such as endotoxin (LPS), phorbol esters, and cytokines including IL-1 and TNF itself (19C22). Although LPS is the major inducer of TNF in infectious disease, in physiological conditions the production of TNF in the bone marrow is mostly induced by inflammatory cytokines. Low concentrations of IL-1 and TNF are, in fact, constitutively produced in the bone marrow (23). Monocyte stimulation with either IL-1 or TNF leads to the activation of the AP-1 family of nuclear proteins (24). The AP-1 family comprises a group of inducible proteins that bind to a common test or 1-way ANOVA, as appropriate. Subsequent mean comparison tests were performed by the Fisher protected least significant difference test. Results RAW 264.7 cells express functional ERs and mRNA for both ER and ER. To investigate whether RAW 264.7 cells, a murine monocytic line known to produce TNF in response to a variety of stimuli (47), express functional ERs, transient transfection experiments were conducted using a SFN reporter plasmid that R788 (Fostamatinib) contained a CAT gene under the transcriptional control of an ERE driven by an SV-40 promoter. Three replicate experiments revealed that 24-hour E2 (10C8 M) treatment increases CAT activity 6-fold (Figure ?(Figure1a),1a), thus demonstrating that RAW 264.7 cells express functional ER. Open in a separate window Figure 1 RAW 264.7 cells express functional ERs and mRNA for ER and ER. (a) Cells were transfected with a reporter plasmid that contained a CAT gene under the transcriptional control of an ERE driven by an SV-40 promoter, and were treated with either E2 or control vehicle. CAT activity was normalized to -galactosidase activity to correct for variability in transfection efficiency and expressed as normalized CAT activity. Mean SEM of.