Michelle L. Key

Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo.

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma‐induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.

Inhibition of osteoblast function in vitro by aminobisphosphonates

Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations ≥100 nM and was strongly toxic at 10 µM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of ‘trabecular’ mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses (≥1 and ≥10 µM, respectively). Short‐term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1–10 µM) was sufficient to inhibit bone mineralisation by ≥85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations ≥100 nM and soluble collagen production at concentrations ≥1 µM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone‐like mineralised substrate, observing similar inhibitory effects, although at concentrations 10–100‐fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non‐toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations ≥1 µM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation. J. Cell. Biochem. 106: 109–118, 2009.

The regulation of osteoblast function and bone mineralisation by extracellular nucleotides: The role of p2x receptors

Extracellular nucleotides, signalling through P2 receptors, regulate the function of both osteoblasts and osteoclasts. Osteoblasts are known to express multiple P2 receptor subtypes (P2X2,5,7 and P2Y(1),(2,4,6)), levels of which change during differentiation. ATP and UTP potently inhibit bone mineralisation in vitro, an effect mediated, at least in part, via the P2Y(2) receptor. We report here that primary rat osteoblasts express additional, functional P2 receptors (P2X1, P2X3, P2X4, P2X6, P2Y(12), P2Y(13) and P2Y(14)). Receptor expression changed with cellular differentiation: e.g., P2X4 receptor mRNA levels were 5-fold higher in mature, bone-forming osteoblasts, relative to immature, proliferating cells. The rank order of expression of P2 receptor mRNAs in mature osteoblasts was P2X4>>P2Y(1)>P2X2>P2Y(6)>P2X1>P2Y(2)>P2Y(4)>P2X6>P2X5>P2X7>P2X3>P2Y(14)>P2Y(13)>P2Y(12). Increased intracellular Ca(2+) levels following stimulation with P2X-selective agonists indicated the presence of functional receptors. To investigate whether P2X receptors might also regulate bone formation, osteoblasts were cultured for 14days with P2X receptor agonists. The P2X1 and P2X3 receptor agonists, α,β-meATP and β,γ-meATP inhibited bone mineralisation by 70% and 90%, respectively at 1μM, with complete abolition at ≥25μM; collagen production was unaffected. Bz-ATP, a P2X7 receptor agonist, reduced bone mineralisation by 70% and 99% at 10μM and 100μM, respectively. Osteoblast alkaline phosphatase activity was similarly inhibited by these agonists, whilst ecto-nucleotide pyrophosphatase/phosphodiesterase activity was increased. The effects of α,β-meATP and Bz-ATP were attenuated by antagonists selective for the P2X1 and P2X7 receptors, respectively. Our results show that normal osteoblasts express functional P2X receptors and that the P2X1 and P2X7 receptors negatively regulate bone mineralisation.

The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels

Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05–0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1–100 μM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1–10 μM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1–10 μM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone.

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation.

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.

Acidosis is a key regulator of osteoblast ecto-nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity.

Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1–6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P < 0.001). Release of ATP, one of the key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5‐fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. J. Cell. Physiol. 230: 3049–3056, 2015.