Figure 2. Cell autonomous stimulation of cAMP by PTHrP in OS.

(A) Cartoon of PTHrP-PTHR1-cAMP-CREB1 axis. (B) qPCR expression of Pthlh normalized to β2m; mean ± SEM (n=3). (C) cAMP levels after anti-PTHrP antibody treatment for fibroblastic OS (light grey) and osteoblastic OS (dark grey). Expressed as normalized mean cAMP ± SEM ((n=3/subtype). (D) Knockdown of Pthlh transcript using 2 independent shRNA (A and B) in indicated OS subtypes. Data normalized to β2m, expressed as mean ± SEM (n=3/subtype). (E) Fold reduction of cAMP levels in sh-Pthlh infected OS subtype cells. IBMX in all treatments, data displayed as normalized mean cAMP ± SEM (n=3/subtype). The data is the mean of 3 independent cell cultures for each subtype. (F) pCREB1/CREB1 protein levels following knockdown of PTHrP. Pan-ACTIN/ATF-1 used as a loading control. Data are representative of 2 independent cell cultures from each OS subtype. (G) Expression of indicated CREB1 target gene transcripts following Pthlh knockdown. Means ± SEM (n=3/subtype). (H) AnnexinV/7-AAD staining of indicated cells following infection with two independent sh-Pthlh (A and B) or sh-Luc control. (I) Quantitation of dead cells in indicated cell type. The data represents 3 independent cell cultures for each type, mean ± SEM (n=3). *p<0.05, **p<0.001, ***p<0.0001. (J) In vivo bilateral grafts of independent fibrobastic OS lines OS80 and 494H with control (sh-Luc) on one flank and sh-Pthlh_A on the other flank. Data expressed as mean weight ± SEM (n=3 tumours per shRNA per cell line; performed once); P value as indicated. See Figure 2—figure supplement 1 and Figure 2—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.13446.006

Figure 2—figure supplement 1. PTHrP, an endogenous ligand for cAMP signaling in OS.

(A) Average number of reads (TMM normalised, RNA-seq) representing the GPCRs expressed in fibroblastic and osteoblastic OS. (B) cAMP assays in 3 independent cell cultures within 2 OS subtypes depicting the effect of PTHrP blockade using anti-PTHrP antibody. (C) cAMP assays in 3 independent cell cultures within 2 OS subtypes post Pthlh knockdown. (D) Western blot showing the loss of PTHrP protein upon knockdown as compared to control cells (E) Cell proliferation post knockdown of Pthlh in two fibroblastic OS cultures. (F, G) Expression of genes in fibroblastic OS cultures (OS80 and 494H) by qPCR and normalized to Hprt. Data expressed as relative expression.

DOI: http://dx.doi.org/10.7554/eLife.13446.007

Figure 2—figure supplement 2. PTHrP overexpression alone does not initiate OS.

(A) Quantification of PTHrP levels in the media of primary osteoblasts infected with control retrovirus (MSCV_Control; empty vector) or PTHrP expressing retrovirus (MSCV_PTHrP). Media from infected cells was applied to UMR106.01 cells and cAMP levels measured by radioimmunoassay. (B) Representative AnnexinV/7-AAD staining profiles of primary osteoblasts following infection with control or PTHrP overexpressing retrovirus. (C) Quantitation of cell death from AnnexinV/7-AAD staining following infection with control or PTHrP overexpressing retrovirus. Data expressed as mean ± SEM, Students t-test was used, *p<0.05, **p<0.001, ***p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.13446.008