A. Barbara

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

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‐1β and TNF‐α 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.

Essential Role of Nuclear Factor of Activated T Cells (NFAT)-mediated Wnt Signaling in Osteoblast Differentiation Induced by Strontium Ranelate

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as β-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.

Strontium ranelate rebalances bone marrow adipogenesis and osteoblastogenesis in senescent osteopenic mice through NFATc/Maf and Wnt signaling

With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt‐ and nuclear factor of activated T‐cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age‐related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.

Age-related decrease in the responsiveness of rat articular chondrocytes to EGF is associated with diminished number and affinity for the ligand of cell surface binding sites

The effect of age on the responsiveness of articular chondrocytes (AC) to epidermal growth factor (EGF) was examined. Cells were isolated by digesting cartilage fragments from the humeral and femoral heads of 21-day old, 8- and 14-month old rats with collagenase. The cells were cultured under standard conditions, as monolayers. DNA synthesis was measured by [3H]thymidine incorporation and cell proliferation by the DNA content of subconfluent cultures. [125I]EGF binding and the amounts of EGF and EGF-receptor mRNAs were determined using confluent cells. DNA synthesis was decreased with age of animals. EGF stimulated DNA synthesis in cultures in 1- and 8-month old rats at low serum concentrations (< 5%), and in cultures in 14-month old animals at high serum concentrations. It also increased 5-day DNA content of cultures compared to serum-treated controls but this effect was weak in cultures in 14-month old rats. The number of high affinity binding sites for [125I]EGF decreased from 37,800 in the 1-month old to 1950 in the 14-month old rat AC. The apparent dissociation constant (Kd) also decreased with age: 0.18 nmol/l in the 1-month old; 0.12 nmol/l in the 8-month old; and 0.07 nmol/l in the 14-month old cells. AC in older rats contained more EGF mRNA and less EGF-receptor mRNA. Incubation of the cells with EGF resulted in down regulation of the EGF- and upregulation of EGF-receptor mRNA expressions. These findings show the age-related quantitative and qualitative alterations in EGF and EGF-receptor which may account, at least in part, for the diminished responsiveness of senescent AC to EGF.

Basal and induced nitric oxide and cGMP productions are decreased in senescent cultured rat articular chondrocytes

The effects of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharide (LPS) on the productions of nitric oxide (NO) and cGMP by cultured articular chondrocyte (AC) monolayers from 1-, 8- and 18-month old male Wistar rats were studied. It was found that basal NO and cGMP productions decrease with the age of animals. The productions were more than 2-fold greater in cells from 1-month old rats then in cells from older animals. IL-1, TNF-alpha, and LPS stimulated all three types of cells to produce NO and cGMP in a time- and concentration-dependent manner. Although the cells from young animals produced more NO per microg DNA, the older counterparts were more sensitive to these agents since they produced more NO upon stimulation then the corresponding non-stimulated controls. At the concentration of 10(-3) M, the nitric oxide synthase (NOS) inhibitor, Ng-monomethyl-L-arginine (L-NMA), blocked, although incompletely, both the basal and stimulated NO and cGMP productions in cells from the 1 and 8-month old rats and only induced productions in 18-month old counterparts. These results show a decreased capacity of unstimulated- and stimulated-AC from old rats to produce NO and cGMP in culture, which may affect the ageing cells in some yet unknown way.

The mechanism of inhibition of endothelin-1-induced stimulation of DNA synthesis in rat articular chondrocytes

Endothelin-1 (ET-1) is a potent mitogen for rat articular chondrocytes (AC) in short term culture (24 h). Prolonged incubation (72 h) of AC with ET-1 resulted in inhibition of [3H]thymidine incorporation. This inhibition seemed to be mediated by prostaglandins (PGs) released in response to ET-1, since indomethacin (INDO) enhanced ET-1-induced [3H]thymidine incorporation. In agreement with this hypothesis, exogenous prostaglandins (PGE2, PGF2alpha and TxB2) blocked all basal, ET-1-induced and ET-1 induced-INDO-enhanced [3H]thymidine incorporation and ET-1 stimulated PGE2 release in a time and concentration-dependent manner. INDO also blocked cGMP production and 6-anilino-5,8-quinolinedione, a relatively specific inhibitor of cGMP formation, enhanced the stimulation and suppressed the inhibition of ET-1-induced DNA synthesis. In addition, 8-bromo-cGMP, an analogue of cGMP, blocked at all time periods studied, both basal and ET-1-induced incorporations of [3H]thymidine. Thus, PGs produced in response to ET-1 counteract the ET-1-induced stimulation of [3H]thymidine incorporation into rat AC by increasing cGMP production.

Endothelin-1 receptors on cultured rat articular chondrocytes: regulation by age, growth factors, and cytokines, and effect on cAMP production.

The presence of endothelin-1 receptor proteins and the expression of their specific mRNAs were studied using 1st passage confluent monolayers of articular chondrocytes, isolated from 1-month and 18-month-old rats following 24-h incubation with several growth factors and cytokines. The ET-1- binding sites were predominantly of ETA subtype since BQ123, but not IRL1038 (ETB receptor subtype agonist), effectively blocked 125I-ET-1 binding. The 18-month-old rat cell monolayers bear approximately twice as many 125I-ET-1-binding sites as the 1-month-old rat cells. PDGF, EGF, and IGF-1 increased the number of binding sites in a concentration-dependent manner in both old and young rat cells with PDGF being the most active and EGF more active than IGF-1. IL-1beta, more potently than LPS, increased the number of binding sites in young rat cells only, whereas b-FGF, TGF-beta and GM-CSF had no effect or decreased slightly 125I-ET-1 binding in both types of cells. TNF-alpha strongly decreased the number of binding sites on both young and old rat cells, only. RT-PCR showed an increased expression of the specific ETA mRNA with the age of animals and in the presence of 50 ng/ml PDGF BB only. The incubation of the cells with ETs 1-3 for 10 min resulted in a 50% decrease of cellular cAMP but the blocking of the receptors with BQ123 prior to their exposure to ETs had no effect on cAMP production whereas IRL1038 counteracted this effect only marginally. This suggests a receptor-independent mechanism for ETs-induced inhibition of cAMP production. However, a 10-min co-incubation of cells with ET-1 and with one of the following agents: cholera toxin, pertussis toxin, indomethacin, L-NMA, U73122 and calphostin resulted in an almost complete (calphostin) or partial suppression of ET-1-induced inhibition of cAMP production. The significance of these findings is unclear but the increased density of ET-1 binding sites on old rat cells and its regulation by certain growth factors or cytokines suggest the involvement of ET-1 in aging and possibly in age-related diseases.