A Novel Cation-Sensing Mechanism in Osteoblasts Is a Molecular Target for Strontium

Abstract

Defining the molecular target for strontium in osteoblasts is important for understanding the anabolic effects of this cation on bone. The current studies demonstrate that a G-protein-mediated response to strontium persists in osteoblasts that lack CASR, suggesting a predominant role for a novel cation-sensing receptor in mediating the osseous response to strontium.Introduction: Strontium has anabolic effects on bone and is currently being developed for the treatment of osteoporosis. The molecular target for strontium in osteoblasts has not been determined, but the existence of CASR, a G-protein-coupled receptor calcium-sensing receptor, raises the possibility that strontium actions on bone are mediated through this or a related receptor. Materials and Methods:We used activation of a transfected serum response element (SRE)-luciferase reporter in HEK-293 cells to determine if CASR is activated by strontium. In addition, we examined strontium-mediated responses in MC3T3-E1 osteoblasts and osteoblasts derived from wild-type and CASR null mice to determine if other cation-sensing mechanisms are present in osteoblasts. Results and Conclusions:We found that strontium stimulated SRE-luc activity in HEK-293 cells transfected with full-length CASR but not in cells expressing the alternatively spliced CASR construct lacking exon 5. In contrast, we found that MC3T3-E1 osteoblasts that lack CASR as well as osteoblasts derived from CASR null mice respond to millimolar concentrations of strontium. The response to strontium in osteoblasts was nonadditive to a panel of extracellular cations, including aluminum, gadolinium, and calcium, suggesting a common mechanism of action. In contrast, neither the CASR agonist magnesium nor the calcimimetic NPS-R568 activated SRE activity in osteoblasts, but the response to these agonists was imparted by transfection of CASR into these osteoblasts, consistent with the presence of distinct cation-sensing mechanisms. Co-expression of the dominant negative G␣q(305-359) minigene also inhibited cation-stimulated SRE activity in osteoblasts lacking known CASR. These findings are consistent with strontium activation of a novel G␣q-coupled extracellular cation-sensing receptor in osteoblasts with distinct cation specificity.

Osteoblast calcium‐sensing receptor has characteristics of ANF/7TM receptors

“…Human embryonic kidney HEK-293 cells were obtained from American Type Culture Collection, Rockville, MD. HEK-293 cells and MC3T3-E1 cells stably expressing rat CASR were created as previously described [Spurney et al, 1999;Pi et al, 2002;Pi and Quarles, 2004]. TmOb (CASR +/+ ) and TmOb (CASR −/− ) are mouse immortalized osteoblastic cell lines derived from wild-type and CASR gene knockout mouse calvaria, respectively, as previously described [Pi et al, 2000].…”

“…In osteoclasts, the prototypic calciumsensing receptor, CASR, a channel, and a recently described cell surface Type II ryanodine receptor are proposed mechanisms for cation-sensing [Zaidi et al, 1989[Zaidi et al, , 1991[Zaidi et al, , 1995Pollak et al, 1993;Sugimoto et al, 1993;Kameda et al, 1998;Kanatani et al, 1999;Seuwen et al, 1999]. In osteoblasts, some studies purport that CASR is responsible for the calcium-sensing in osteoblasts [Nemeth and Scarpa, 1987;Brown et al, 1993;House et al, 1997;Kameda et al, 1998;Yamaguchi et al, 1998aYamaguchi et al, ,c,e, 2001Kanatani et al, 1999;Mathas et al, 2001;Brown, 2003;Chattopadhyay et al, 2004;Dvorak et al, 2004], whereas other studies provide evidence for the presence of a novel calcium-sensing receptor in osteoblasts, called Ob.CASR, and possible redundant calcium-sensing mechanisms in bone [Pi and Quarles, 2004].…”

“…First, CASR knockout mice fail to display a bone phenotype in the absence of hyperparathyroidism [Garner et al, 2001;Tu et al, 2003] and the absence of CASR does not alter either the responsive of osteoblasts to extracellular calcium or their ability to undergo the typical maturational program leading to the formation of mineralized extracellular matrix ex vivo [Pi et al, 2000]. In addition, several laboratories have failed to confirm the expression of CASR in osteoblasts Pi et al, 1999;Shalhoub et al, 2003;Pi and Quarles, 2004]. It has been shown that CASR expression can be lost in cell cultures; consequently the absence of CASR in the clonal MC3T3-E1 cell lines does not preclude its presence in primary cultures [House et al, 1997;Kameda et al, 1998;Yamaguchi et al, 1998a,c,e;Kanatani et al, 1999].…”

“…It has been shown that CASR expression can be lost in cell cultures; consequently the absence of CASR in the clonal MC3T3-E1 cell lines does not preclude its presence in primary cultures [House et al, 1997;Kameda et al, 1998;Yamaguchi et al, 1998a,c,e;Kanatani et al, 1999]. However, distinct calciumsensing responses have been detected in osteoblastic cultures lacking CASR [Demarest et al, 1997;Mailland et al, 1997;Quarles et al, 1997;Bapty et al, 1998;Pi et al, 1999;Shalhoub et al, 2003;Pi and Quarles, 2004], suggesting the presence of another receptor.…”

“…Also, the divalent strontium cation stimulates osteoblast-mediated bone formation and inhibits osteoclast-meditated bone resorption in preclinical studies [Delannoy et al, 2002;Marie, 2003;Verberckmoes et al, 2003;Barbara et al, 2004] and the drug Strontium Ranelate is a recently approved treatment of osteoporosis [Meunier et al, 2004]. The predominant mechanism of strontium on bone is uncertain, since strontium can activate CASR [Brown, 2003;Coulombe et al, 2004], as well as Ob.CASR [Pi and Quarles, 2004] in vitro.…”