Figure 3. Persistent, elevated cAMP production in OS compared to primary osteoblasts.

(A) cAMP levels in indicated cells (1000 cells per well) with and without IBMX treatment. Data from 3 independent cultures per type, mean ± SEM. (B) Intracellular cAMP levels in indicated cell type following treatment with forskolin; mean ± SEM (n=3 per cell type; 1000 cells per well); statistical significance for OS vs normal Ob; all points of fibroblastic vs osteoblastic OS not significantly different. (C) Western blot and (D) quantification of CREB1/pCREB1 during a time course of cAMP activation by forskolin. Data representative of 2 independent cultures each. (E) Heat map of qPCR data. CREB1 target gene expression in indicated cells; data expressed as relative expression. (F) ChIP analysis of CREB1/pCREB1 on the promoters of indicated genes over a 2 hr time course following stimulation with forskolin. Data is represented as percentage of input. The data from 2 independent cell lines for each subtypes mean occupancy ± SEM (n=2–3 assays per line). (G) Western blot of CREB1/pCREB1 expression in proliferating non stimulated cultures, β-ACTIN used as a loading control. Data representative of 3–4 independent cell lines from each type. (H) CREB1 transcript expression in human osteoblasts and osteosarcoma (data taken from PMID: 25961939). (I) Western CREB1/pCREB1 expression in indicated cell types under differentiative conditions, ATF-1 used as a loading control. (J) Relative expression of negative regulators of cAMP in OS subtypes compared to primary osteoblasts by qPCR and normalized to β2m represented as a heat map (n=3/cell type). *p<0.05, **p<0.001, ***p<0.0001. See Figure 3—figure supplement 1 and Figure 3—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.13446.009

Figure 3—figure supplement 1. cAMP is constitutive in mouse OS leading to continuous phosphorylation of CREB1.

(A) Quantification of cAMP in primary osteoblasts, and OS with and without IBMX treatment (500 cells/well). Data represents 3 independent cell cultures for each type, mean ± SEM. (B) Kinetics of cAMP accumulation in OS subtypes as compared to primary osteoblasts in the absence of IBMX treated with 10 μM forskolin (using 500 cells/well). (C-D) qPCR validation of CREB1 target gene expression following 10 μM forskolin treatment over a time course of 2 hr. The data represents 3 independent cell cultures for each subtype ± SEM (n=3). (E) Expression of CREB1 target genes post knockdown of Creb1 using siRNA by qPCR and normalized to β2m. Means ± SEM (n=3). The data represents 3 independent cell lines for each subtype ± SEM (n=3). Effect of Creb1 knockdown on Crem1 expression in fibroblastic OS (F) and osteoblastic OS (G), respectively. Expression of Crem1 by qPCR and normalized to β2m. Means ± SEM (n=3). (H) Expression of Creb1 in primary osteoblasts compared to each OS subtype by qPCR and normalized to β2m. Means± SEM (n=3). For all the experiments above Students t-test was used to assess statistical significance, *p<0.05, **p<0.001, ***p<0.0001

DOI: http://dx.doi.org/10.7554/eLife.13446.010

Figure 3—figure supplement 2. Altered Creb1 dynamics in osteoblasts and OS.

(A) Expression of Creb1 during in vitro differentiation of primary osteoblasts, data expressed as mean ± SEM (n=3). (B) Expression of Creb1 during in vitro differentiation of fibroblastic OS, data expressed as mean ± SEM (n=3). (C) Expression of CREB1 protein during in vitro differentiation of primary human osteoblasts isolated from normal healthy donors (17–35 year old). (D) Expression of CREB1 transcript during in vitro differentiation of primary human osteoblasts and markers of osteoblast differentiation state as indicated, data expressed as normalized gene expression compared to β2microglobulin expression; graphed as mean ± SEM (n=5 independent donor samples). (E) Expression of negative regulators of cAMP in OS subtypes compared to primary osteoblasts by qPCR and normalized to β2m (n=3/cell type). *p<0.05, **p<0.001, ***p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.13446.011