Figure 6: IGFBP5 effects on osteoblasts and osteoclasts. | Nature Communications

Figure 6: IGFBP5 effects on osteoblasts and osteoclasts.

From: c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling

Figure 6

(a) Mouse calvarial osteoblasts were cultured at subconfluence and treated with the indicated concentrations of soluble IGFBP5 for 30 min. Western blot analysis of c-Src activating phosphorylation and total c-Src protein, normalized versus actin. (b) Mouse calvarial osteoblasts were cultured at overconfluence and treated with the indicated concentrations of soluble IGFBP5 for 30 min. Western blot analysis of c-Src activating phosphorylation and total c-Src protein, normalized versus actin. (c) ELISA assay for mIGFBP5 detection in the conditioned media from sub- and overconfluent osteoblasts treated with 5 ng ml−1 rhIL-6 or with 2 μM PP1 (mean±s.d., n=3. *P<0.05 and **P<0.005, Student's t-test). (d) Osteoclast precursors were differentiated in the presence of 50 ng ml−1 M-CSF, 120 ng ml−1 RANKL and the indicated concentrations of rhIGFBP5. Tartrate-resistant acid phosphatase (TRAcP) staining. Scale bar, 50 μm. (e) Count of TRAcP-positive multinucleated cells per well (mean±s.d., n=4, **P<0.0002; *P<0.04, Student's t-test) in the cultures shown in d. (f) Osteoclasts obtained by M-CSF/RANKL treatment were detached and replated on bone sections in the presence of the indicated IGFBP5 concentrations to evaluate bone resorption by the pit index (mean±s.d., n=3, *P<0.05, Student's t-test). (g) Sub- and overconfluent osteoblasts were treated with the indicated concentrations of IGFBP5 per 24 h. RT–PCR and relative quantification of Rankl and Opg expression, normalized to Gapdh, and their expression ratio (mean±s.d., n=3, *P<0.05; **P<0.005; #P<0.01, Student's t-test). (h) Cartoon describing the amplification and inhibitory loops involving c-Src, IL-6 and IGFBP5 in immature osteoblasts, and the IGFBP5-dependent osteoblast-to-osteoclast cross-talk. (i) Cartoon describing the relationship between c-Src, Runx2 and IGFBP5 in mature osteoblasts, and the IGFBP5-dependent osteoblast-to-osteoclast cross-talk. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

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