Calcium sensing receptor-dependent and receptor-independent activation of osteoblast replication and survival by strontium ranelate

Abstract

Age-related osteopenia is characterized by a negative balance between bone resorption and formation. The anti-osteoporotic drug strontium ranelate was found to reduce bone resorption and to promote bone formation. Here, we investigated the implication of the calcium-sensing receptor (CaSR) in the response to strontium ranelate using osteoblasts from CaSR knockout [CaSR(-/-)] and wild-type [CaSR(+/+)] mice. We showed that calcium and strontium ranelates increased cell replication in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts. Strontium ranelate rapidly increased ERK1/2 phosphorylation in [CaSR(+/+)] but not in [CaSR(-/-)] osteoblasts, indicating that strontium ranelate can act independent of the CaSR/ERK1/2 cascade to promote osteoblast replication. We also showed that strontium ranelate prevented cell apoptosis induced by serum deprivation or the pro-inflammatory cytokines IL-1beta and TNF-alpha in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts, indicating that CaSR is not the only receptor involved in the protective effect of strontium ranelate on osteoblast apoptosis. Strontium ranelate activated the Akt pro-survival pathway in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts, and pharmacological inhibition of Akt abrogated the anti-apoptotic effect of strontium ranelate. Furthermore, both the proliferative and anti-apoptotic effects of strontium ranelate in [CaSR(-/-)] and [CaSR(+/+)] osteoblasts were abrogated by selective inhibition of COX-2. The results provide genetic and biochemical evidence that the effects of strontium ranelate on osteoblast replication and survival involve ERK1/2 and Akt signalling and PGE2 production, independent of CaSR expression. The finding that CaSR-dependent and CaSR-independent pathways mediate the beneficial effects of strontium ranelate on osteoblasts, provides novel insight into the mechanism of action of this anti-osteoporotic agent on osteoblastogenesis.

Figure 1

Calcium chloride and strontium ranelate (SrRan) stimulate osteoblast replication independent of CaSR expression. (A) Immunoprecipitation analysis showing expression of CaSR in [CaSR^+/+] but not [CaSR^−/−] osteoblasts isolated from newborn mice. Protein lysates were immunoprecipitated with anti‐CaSR, electrophoresed, reacted with CaSR antibody and immunoblots were probed with a peroxidase‐coupled specific secondary antibody. (B, C) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were treated with calcium chloride or SrRan at the indicated concentration for 24 hrs. (D, E) Time‐dependent increase in cell replication in [CaSR^+/+] and [CaSR^−/−] osteoblasts. After 24–48 hrs of incubation with calcium or SrRan, cells were pulsed with BrdU and processed by ELISA to determine BrdU incorporation. Results are expressed as mean ± SD (n= 6). * indicates a significant difference with untreated controls (P < 0.05).

Figure 2

Strontium ranelate (SrRan) rapidly induces ERK1/2 phosphorylation in [CaSR^+/+] but not [CaSR^−/−] osteoblasts. (A, B) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were treated with SrRan or ‘calcium chloride’ at the indicated concentrations for 10 min., and ERK1/2 phosphorylation was determined by Western blot analysis. (C, D) Time‐course effect of SrRan on ERK1/2 phosphorylation in osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice. The gels were scanned and the levels were corrected for loading using β‐actin. The data are expressed as treated over control ratio (T/C).

Figure 3

Strontium ranelate (SrRan) protects against apoptosis in osteoblasts. (A, B) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were cultured in DMEM supplemented with 5% foetal calf serum (FCS) or FCS‐deprived (1% FCS) medium and treated with SrRan at the indicated concentration for 24 hrs. (C, D) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice cultured were treated with TNF‐α or IL‐1‐β (5 ng/ml) in the presence of SrRan at the indicated concentrations for 24 hrs. Cell apoptosis was evaluated by the Apopercentage assay. Results are expressed as mean ± SD (n= 6). a and b indicate a significant difference with the corresponding 5% and 1% FCS control groups, respectively. c and d indicate a significant difference with the corresponding untreated control groups, respectively (P < 0.05).

Figure 4

Strontium ranelate (SrRan) activates Akt signalling in osteoblasts. Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were cultured in DMEM supplemented with 5% FCS for 24 hrs, then treated with or without increasing doses of SrRan at the indicated concentrations for 10 (A, B) or 30 min. (C, D). Phosphorylation level of Akt was evaluated by Western blot analysis, relative quantification was performed after correction for β‐actin content, and the results expressed as treated over control ratio (T/C).

Figure 5

The anti‐apoptotic effect of strontium ranelate (SrRan) is mediated by PI3K Akt signalling in osteoblasts. (A, B) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were cultured in DMEM supplemented with 10% FCS or 1% FCS for 24 hrs and treated with or without SrRan for 24 hrs. Effector caspases activity was determined and the results are expressed as treated over control ratio (T/C) (mean ± SD). * indicates a significant difference with the 1% FCS control group (P<0.05). (C, D) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were cultured in DMEM supplemented with 1% FCS, for 24 hrs, treated with or without SrRan (3 mM) for 24 hrs in the presence or absence of the PI3K inhibitor wormanin (10 mM), and effector caspases activity was determined. Results are expressed as mean ± SD (n= 6). a and b indicate a significant difference with the 1% FCS and SrRan groups, respectively (P < 0.05).

Figure 6

The effects of strontium ranelate (SrRan) in osteoblasts involve prostaglandins. (A, B) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were treated with SrRan (3 mM) for 24 hrs in the presence of the COX‐2 inhibitor NS‐398 (0.1 μM) or the solvent, cells were pulsed with BrdU and processed by ELISA to determine BrdU incorporation. Results are expressed as treated over control ratio (T/C) (mean ± SD; n= 6). a and b indicate a significant difference with untreated and SrRan‐treated cells, respectively (P < 0.05). (C, D) Osteoblasts from [CaSR^+/+] and [CaSR^−/−] mice were cultured in DMEM supplemented with 5% FCS (control) or 1% FCS for 24 hrs and treated with SrRan (3 mM) for 24 hrs in the presence of the COX‐2 inhibitor NS‐398 (0.1 μM) or the solvent, and cell apoptosis was evaluated by the Apopercentage assay. Results are expressed as treated over control ratio (T/C) (mean ± SD; n= 6). a, b and c indicate a significant difference with the 10% FCS, 1% FCS and SrRan‐treated groups, respectively (P < 0.05).

Figure 7

Proposed mechanisms by which strontium ranelate (SrRan) controls cell replication and protects against apoptosis in murine primary osteoblasts. SrRan activates cell replication in part through CasR‐mediated ERK1/2 signalling and another unidentified cation‐sensing receptor. In addition, SrRan induces anti‐apoptotic effects in murine osteoblasts through CaSR‐dependent and CaSR‐independent mechanisms involving the PI3K Akt pro‐survival pathway.