The Transient Receptor Potential Ion Channel TRPV6 Is Expressed at Low Levels in Osteoblasts and Has Little Role in Osteoblast Calcium Uptake

Background: TRPV6 ion channels are key mediators of regulated transepithelial absorption of Ca2+ within the small intestine. Trpv6-/- mice were reported to have lower bone density than wild-type littermates and significant disturbances in calcium homeostasis that suggested a role for TRPV6 in osteoblasts during bone formation and mineralization. TRPV6 and molecules related to transepithelial Ca2+ transport have been reported to be expressed at high levels in human and mouse osteoblasts. Results: Transmembrane ion currents in whole cell patch clamped SaOS-2 osteoblasts did not show sensitivity to ruthenium red, an inhibitor of TRPV5/6 ion channels, and 45Ca uptake was not significantly affected by ruthenium red in either SaOS-2 (P = 0.77) or TE-85 (P = 0.69) osteoblastic cells. In contrast, ion currents and 45Ca uptake were both significantly affected in a human bronchial epithelial cell line known to express TRPV6. TRPV6 was expressed at lower levels in osteoblastic cells than has been reported in some literature. In SaOS-2 TRPV6 mRNA was below the assay detection limit; in TE-85 TRPV6 mRNA was detected at 6.90±1.9 × 10−5 relative to B2M. In contrast, TRPV6 was detected at 7.7±3.0 × 10−2 and 2.38±0.28 × 10−4 the level of B2M in human carcinoma-derived cell lines LNCaP and CaCO-2 respectively. In murine primary calvarial osteoblasts TRPV6 was detected at 3.80±0.24 × 10−5 relative to GAPDH, in contrast with 4.3±1.5 × 10−2 relative to GAPDH in murine duodenum. By immunohistochemistry, TRPV6 was expressed mainly in myleocytic cells of the murine bone marrow and was observed only at low levels in murine osteoblasts, osteocytes or growth plate cartilage. Conclusions: TRPV6 is expressed only at low levels in osteoblasts and plays little functional role in osteoblastic calcium uptake

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Osteoclasts, the multinucleated bone-resorbing cells, arise through fusion of precursors from the myeloid lineage. However, not all osteoclasts are alike; osteoclasts at different bone sites appear to differ in numerous respects. We investigated whether bone marrow cells obtained from jaw and long bone differed in their osteoclastogenic potential. Bone marrow cells from murine mandible and tibiae were isolated and cultured for 4 and 6 days on plastic or 6 and 10 days on dentin. Osteoclastogenesis was assessed by counting the number of TRAP+ multinucleated cells. Bone marrow cell composition was analyzed by FACS. The expression of osteoclast- and osteoclastogenesis-related genes was studied by qPCR. TRAP activity and resorptive activity of osteoclasts were measured by absorbance and morphometric analyses, respectively. At day 4 more osteoclasts were formed in long bone cultures than in jaw cultures. At day 6 the difference in number was no longer observed. The jaw cultures, however, contained more large osteoclasts on plastic and on dentin. Long bone marrow contained more osteoclast precursors, in particular the myeloid blasts, and qPCR revealed that the RANKL:OPG ratio was higher in long bone cultures. TRAP expression was higher for the long bone cultures on dentin. Although jaw osteoclasts were larger than long bone osteoclasts, no differences were found between their resorptive activities. In conclusion, bone marrow cells from different skeletal locations (jaw and long bone) have different dynamics of osteoclastogenesis. We propose that this is primarily due to differences in the cellular composition of the bone site-specific marrow

Voltage sensitivity of the osteoclast calcium receptor.

We demonstrated previously that osteoclasts possess a divalent cation-sensitive "receptor", the Ca2+ receptor. Activation of the Ca2+ receptor by the surrogate cation Ni2+ was shown to elicit an increase in cytosolic [Ca2+] to a peak value followed by an exponential decline. In the present study we examined the influence of surface membrane voltage on the kinetics of Ca2+ receptor inactivation. The K+ ionophore, valinomycin was applied to intercept the declining phase of the cytosolic [Ca2+] transient elicited by application of between 50 microM- and 5 mM-[Ni2+]. This resulted in a sustained elevation of cytosolic [Ca2+] or even a 'hump' followed by a gradual decline. Such a kinetic alteration persisted in a Ca(2+)-free solution, but was abolished in high extracellular [K+] (105 mM). Thus, we demonstrate for the first time to our knowledge, a modulatory effect of membrane potential on the function of the osteoclast Ca2+ receptor

Voltage sensitivity of the osteoclast calcium receptor.

We demonstrated previously that osteoclasts possess a divalent cation-sensitive "receptor", the Ca2+ receptor. Activation of the Ca2+ receptor by the surrogate cation Ni2+ was shown to elicit an increase in cytosolic [Ca2+] to a peak value followed by an exponential decline. In the present study we examined the influence of surface membrane voltage on the kinetics of Ca2+ receptor inactivation. The K+ ionophore, valinomycin was applied to intercept the declining phase of the cytosolic [Ca2+] transient elicited by application of between 50 microM- and 5 mM-[Ni2+]. This resulted in a sustained elevation of cytosolic [Ca2+] or even a 'hump' followed by a gradual decline. Such a kinetic alteration persisted in a Ca(2+)-free solution, but was abolished in high extracellular [K+] (105 mM). Thus, we demonstrate for the first time to our knowledge, a modulatory effect of membrane potential on the function of the osteoclast Ca2+ receptor

Chapter 28 Genetics and cell biology of calcitonin action

Calcitonin is a 32-amino acid circulating hormone involved in skeletal homeostasis. Its primary target cell is the osteoclast, the bone-resorbing cell. Low picomolar concentrations of calcitonin inhibit bone resorption and produce a characteristic morphological change in isolated osteoclasts. The latter change has been separated biophysically into quiescence (Q) and retraction (R) components. Both components are involved in the inhibition of bone resorption, but are nevertheless distinct. The Q effect is mediated by a cholera toxin-sensitive Gs-like G protein involving a cyclic AMP-dependent pathway. The R effect is triggered by a rise of cytosolic [Ca2+] and probably involves the activation of a pertussis toxin-sensitive Gq-like G protein. The parallel activation pathways appear to be coupled proximally to separate calcitonin receptor subtypes. One of these cross-reacts with calcitonin and its related peptides, amylin and calcitonin gene-related peptide, while the other appears highly calcitonin-specific. However, in the renal epithelial cells, there is evidence for differential coupling of a single receptor to more than one transduction pathway. This occurs in a cell cycle-dependent manner. Two members of the calcitonin receptor family, the porcine renal receptor and the human ovarian receptor, have been cloned and sequenced. Both are of a high affinity (nM) and are functionally coupled to increases in intracellular cyclic AMP. The recombinant porcine renal calcitonin receptor also displays coupling to the phospholipase C-IP3 system. Both receptors show little (30%), indicating that the receptors for these hormones represent a new family of G protein-coupled receptors with unique structural and functional properties. © 1998 Elsevier B.V. All rights reserved