Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones

Abstract

We investigated the direct effects of changes in free ionized extracellular calcium concentrations ([Ca 2؉ ]o) on osteoblast function and the involvement of the calcium-sensing receptor (CaR) in mediating these responses. CaR mRNA and protein were detected in osteoblast models, freshly isolated fetal rat calvarial cells and murine clonal osteoblastic 2T3 cells, and in freshly frozen, undecalcified preparations of human mandible and rat femur. In fetal rat calvarial cells, elevating [Ca 2؉ ]o and treatment with gadolinium, a nonpermeant CaR agonist, resulted in phosphorylation of the extracellular signal-regulated kinases 1 and 2, Akt, and glycogensynthase kinase 3␤, consistent with signals of cell survival and proliferation. In agreement, cell number was increased under these conditions. Expression of the osteoblast differentiation markers core binding factor ␣1, osteocalcin, osteopontin, and collagen I mRNAs was increased by high [ (2, 3) and alter the levels of expression of some differentiation markers (4, 5). During mineralization, decreases in [Ca 2ϩ ] o are also likely to occur (6), but the effect of lowering [Ca 2ϩ ] o in bone cells has not been extensively addressed.The mechanism of [Ca 2ϩ ] o -sensing by osteoblasts is unclear. The parathyroid extracellular calcium-sensing receptor (CaR) is a key player in the maintenance of a constant systemic [Ca 2ϩ ] o , predominantly through regulation of parathyroid hormone (PTH) secretion and urinary calcium excretion (7,8). CaR is also present in osteoblasts (9 and references therein), where a functional role is currently debated. Recently two studies have shown that CaRdeficient mice exhibit an essentially normal skeletal phenotype when the hyperparathyroidism resulting from the lack of the parathyroid CaR is prevented (10, 11). Thus, it remains unclear whether the osteoblast CaR is a true regulator of bone function or whether its expression is vestigial (12).In this study, we investigated the effects of both decreasing and increasing [Ca 2ϩ ] o on osteoblast proliferation and intracellular signaling events, the expression of several osteoblast differentiation markers [core binding factor ␣1 (Cbfa1, also termed Runx2 and Osf2), osteocalcin (OC), osteopontin (OP), and type I collagen (collaI)], the activity of alkaline phosphatase (AlP), and mineralized nodule formation in the absence of systemic calciotropic factors, namely PTH and vitamin D. We further investigated the role played by the CaR in these events using an alternative, nonpermeant CaR agonist, gadolinium (Gd 3ϩ ) and a CaR inhibitor, NPS 89636 (a ''calcilytic''). We used well characterized osteoblast models, freshly isolated fetal rat calvarial cells (FRC) (13) and the clonal murine osteoblast cell line, 2T3 cells (14). The expression of CaR in freshly frozen sections of rat and human bone was also determined. Materials and MethodsAnimals. Sprague-Dawley rats (Charles River Breeding Laboratories) were killed by cervical dislocation and used in accordance to the U.K. Animals Scientific Procedures...

Osteal Tissue Macrophages Are Intercalated throughout Human and Mouse Bone Lining Tissues and Regulate Osteoblast Function In Vitro and In Vivo

“…We next demonstrated that macrophages are essential for osteoblast mineralization in response to the physiological proanabolic stimulus of elevated extracellular calcium (28). We demonstrated that both OsteoMacs and BMM were able to promote osteoblast mineralization in response to this stimulus.…”

“…Extracellular calcium is known to be an important physiological stimulus of bone formation (28) and high extracellular calcium concentrations are a characteristic feature of the bone microenvironment (29). The apparent macrophage contribution to osteoblast mineralization in vitro (Fig.…”

Vitamin D and calcium insufficiency-related chronic diseases: molecular and cellular pathophysiology

“…This is difficult to understand because the effect of even large variations in calcium intake levels on extracellular calcium concentrations [Ca 2 þ ] o is attenuated (Dvorak et al, 2004;Brown and Lian, 2008;Chang et al, 2008), as well as in limitation of cellular growth of normal and neoplastic cells (Rodland, 2004). Conversely, low dietary calcium causes hyperparathyroidism by impairment of CaR activity and, by the same token, can be linked to the development of osteoporosis and of various malignancies.…”

Effects of a novel calcium aluminate cement on the early events of the progression of osteogenic cell cultures

“…In addition, because of the dissociation of calcium hydroxide in the culture medium, differences in calcium and hydroxyl concentrations between groups should also take place. Whereas slightly higher extracellular calcium concentration than physiological values has been shown to stimulate osteoblast cell viability, proliferation, differentiation, and function (16), cellular calcium overload can cause cytotoxicity and trigger either apoptotic or necrotic cell death (17). Concerning the hydroxyl ion concentration, its higher levels have been directly correlated with altered extracellular matrix organization, reduced ALP activity, and bone-like nodule formation in osteogenic cell cultures (18).…”