Evidence that conditionally immortalized human osteoblasts express an osteocalcin receptor

• Published in: Bone (New York, N.Y.) N.Y.), 1999-11, Vol.25 (5), p.535-543
• Main Authors: Bodine, P.V.N, Komm, B.S
• Format: Article
• Language: English
• Subjects: Adenylate Cyclase Toxin; Adult; Alkaline phosphatase; Alkaline Phosphatase - antagonists & inhibitors; Alkaline Phosphatase - metabolism; Animals; Biological and medical sciences; Biosensing Techniques; Bone; Cattle; Cell Line, Transformed; Colforsin - pharmacology; Cyclic AMP - biosynthesis; Cyclic AMP - metabolism; Cytosensor; Dose-Response Relationship, Drug; Fundamental and applied biological sciences. Psychology; G-proteins; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; Humans; Mice; Microphysiometry; Osteoblasts; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - enzymology; Osteoblasts - metabolism; Osteocalcin; Osteocalcin - metabolism; Osteocalcin - pharmacology; Osteocalcin - physiology; Pertussis Toxin; Rats; Receptors, Peptide - biosynthesis; Receptors, Peptide - physiology; Signal Transduction - physiology; Skeleton and joints; Tumor Cells, Cultured; Vertebrates: osteoarticular system, musculoskeletal system; Virulence Factors, Bordetella - pharmacology
• ISSN: 8756-3282; 1873-2763
• DOI: 10.1016/S8756-3282(99)00213-6

Osteocalcin (OC) is an abundant noncollagenous bone matrix protein, yet its function is largely unknown. However, targeted ablation of two OC genes in mice lead to increased bone formation (Ducy et al. Nature 382:448–452; 1996). This implied that OC inhibits osteoblast activity, and that these cells express an OC receptor. In order to characterize the putative OC receptor, we used the Cytosensor microphysiometer to measure responses of a proliferative-stage, conditionally immortalized human osteoblast cell line (HOB-03-C5) to purified bovine OC (bOC). The Cytosensor measures a change in the extracellular acidification rate, which is primarily a measurement of metabolic activity. Treatment of the HOB cells for 5–60 sec with 0.17 μmol/L bOC generated a time-dependent, transient increase in the acidification rate that became optimal after 25 sec. Likewise, treatment of the cells for 25 sec with 0.021 to 1.9 μmol/L bOC caused a dose-dependent 70% increase in the acidification rate. Pretreatment of the cells for 2 h with inhibitors of adenylyl cyclase, phospholipase C, and intracellular calcium release inhibited the response of the cells to bOC by 50%–100%, which suggested that the putative OC receptor was coupled to a G-protein. These observations from the Cytosensor were confirmed by measuring intracellular cyclic-adenosine monophosphate (cAMP) concentrations in response to bOC. Treatment of the cells for 10 min with bOC decreased basal cAMP levels by 65% in a dose-dependent manner with an IC 50 of 0.22 μM. However, cotreatment of the cells with forskolin, which activates adenylyl cyclase, blunted this suppression. Moreover, pretreatment of the cells with pertussis toxin for 48 h, which inhibits Gαi proteins, reversed the suppressive effects of bOC on cAMP production. Treatment of the HOB cells for 48 h with 0.19 to 1.5 μmol/L bOC caused a dose-dependent 40% decrease in alkaline phosphatase activity with an IC 50 of 0.21 μmol/L, which suggested that OC may inhibit HOB activity. Finally, although the maturation stage, conditionally immortalized HOB-02-C1 cells also responded to bOC as measured by the Cytosensor, two osteosarcoma cell lines, SaOS-2 and ROS 17/2.8, exhibited a 5- to 10-fold lower response to the bone matrix protein, suggesting that the putative OC receptor was downregulated in these cells. However, all of these bone cell lines responded to parathyroid hormone treatment. In conclusion, these results provide evidence that the HOB cells express an