c-Fos induction by gut hormones and extracellular ATP in osteoblastic-like cell lines.

It is widely accepted that the c-Fos gene has a role in proliferation and differentiation of bone cells. ATP-induced c-Fos activation is relevant to bone homeostasis, because nucleotides that are present in the environment of bone cells can contribute to autocrine/paracrine signalling. Gut hormones have previously been shown to have an effect on bone metabolism. In this study, we used the osteoblastic Saos-2 cell line transfected with a c-Fos-driven reporter stimulated with five gut hormones: glucose inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin and obestatin, in the presence or absence of ATP. In addition, TE-85 cells were used to determine the time course of c-Fos transcript induction following stimulation with GLP-1, and GLP-2 with or without ATP, using reverse transcription qPCR. The significant results from the experiments are as follows: higher level of c-Fos induction in presence of GIP, obestatin (p = 0.019 and p = 0.011 respectively), and GIP combined with ATP (p < 0.001) using the luciferase assay; GLP-1 and GLP-2 combined with ATP (p = 0.034 and p = 0.002, respectively) and GLP-2 alone (p < 0.001) using qPCR. In conclusion, three of the gut peptides induced c-Fos, providing a potential mechanism underlying the actions of these hormones in bone which can be directed or enhanced by the presence of ATP.

Adiponectin receptors are present in RANK-L-induced multinucleated osteoclast-like cells.

Adiponectin is an adipokine that has been related to bone metabolism. Data on adiponectin receptors (AdipoR1, -R2) in osteoclasts have shown discrepancies. In this study we carried out observations of AdipoR1, -R2 in peripheral blood mononuclear cells that were induced to differentiate into osteoclasts. AdipoR1, -R2 were screened using reverse transcription and quantitative PCR and immunofluorescence. Acid phosphatase and Cathepsin-K were evaluated as osteoclastic markers. Results showed that acid phosphatase was expressed from day 1 whereas Cathepsin-K started from day 7. AdipoR1 and -R2 showed expression from day 1, with greater expression for AdipoR1 than AdipoR2. The immunofluorescent patterns were observed in the cells cultured under three different conditions: non-supplemented medium, added M-CSF, or medium with M-CSF, and RANK-L. The non-supplemented control did not display specific fluorescence whereas specific and strong signals were detected in cells cultured with combined M-CSF and RANK-L from day 7. The fluorescence patterns were detected mainly at the periphery of the cells, and in the cytoplasm, showing a localized patchy pattern for both receptors. In contrast, a diffuse fluorescent pattern was detected in the cytoplasm of the cells with M-CSF alone. In summary, AdipoR1 and -R2 were detected by quantitative PCR and immunofluorescence. The immunofluorescence patterns suggest that adiponectin receptors are located, or re-located, in the plasma membrane with distribution in the cytoplasm when mononuclear cells are committed to differentiate to osteoclasts. These findings could be a reasonable explanation for the controversy found in the published literature regarding the role of adiponectin in bone metabolism.