James William Poser

Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids

Cells have been cultured from human bone that possess several characteristics of osteoblasts, including the capacity to produce osteocalcin (bone Gla protein). In these cultures the production of osteocalcin is dependent on 1,25(OH)2D3 but is not affected by 24,25(OH)2D3 either alone or in combination with 1,25(OH)2D3. Two glucocorticoids, prednisolone and deflazacort, reverse the stimulation of osteocalcin synthesis by 1,25(OH)2D3 in a dose-dependent manner (10(-9) - 10(-6)M). Parathyroid hormone also inhibits osteocalcin production in a dose-dependent fashion (0.2-5 IU/ml). These results demonstrate that human bone cell cultures may be of considerable value in investigating the hormonal and pharmacologic regulation of the production of osteocalcin and other bone proteins in vitro.

Chemotactic activity of theγ-carboxyglutamic acid containing protein in bone

SummaryWe have found that theγ-carboxyglutamic acid (GLA)-containing protein from bone (BGP, osteocalcin) has chemotactic activityin vitro for a number of cells which are found adjacent to endosteal bone surfacesin vivo. Using the Boyden chamber technique for measuring cell chemotaxisin vitro, we have shown that BGP is chemotactic for cultured human breast cancer cells, human and mouse monocytes, and for cultured rat osteosarcoma cells which have the characteristics of osteoblasts. The migration of these cells in response to BGP is unidirectional and not due to spontaneous or random migration. A synthetic peptide (Phe-Tyr-Gly-Pro-Val), which is identical to the carboxyterminal peptide cleaved from BGP when digested by trypsin, is also chemotactic for the same cells. BGP retains its chemotactic activity after conversion of theγ-carboxyglutamic acid residues to glutamic acid, indicating that this biological effect requires neitherγ-carboxyglutamate nor the ability of BGP to bind calcium. Since BGP is released from bone during states of increased bone turnover, it is possible that this chemotactic effect of the protein may be a mechanism for recruitment of these cells to sites of active bone remodeling.

The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro

Cultured adherent human mononuclear cells produce factor(s) which stimulate the release of calcium from new-born mouse calvaria in organ culture. This stimulation of bone resorption is accompanied by an inhibition of the incorporation of [3H]proline into collagen which is independent of increased prostaglandin production by the bone. When human osteoblast-like cells are treated with conditioned medium from human mononuclear cells, collagen accounts for a decreased proportion of the protein synthesised. This effect on matrix synthesis is not accompanied by an inhibitory action of the monocyte-conditioned medium preparations on net cell proliferation. In human osteoblast-like cell cultures, partially purified human interleukin 1 also inhibits the production of the bone-specific protein osteocalcin in a dose-dependent fashion. These observations are consistent with the hypothesis that products of human monocytes similar to, or identical with, human interleukin 1 may be important regulators of bone metabolism and may contribute to the bone loss seen in diseases such as chronic rheumatoid arthritis.

Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats

SummaryWe previously reported that pharmacologic doses of 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) given for 2–3 days, inhibited osteoblastic collagen synthesis in young rats. In this study, we tested the effects of 5, 25, and 125 ng of 1,25(OH)2D3 injected subcutaneously into 6-week-old rats for 12 or 18 days. In rats given 125 ng, cortical bone of distal half femurs exhibited decreased calcium (Ca) content but dry weight and hydroxyproline (Hyp) content were no different from control. Trabecular bone Ca was not different from control but dry weight and Hyp were increased. When cortical and trabecular bone were combined, there was a decrease in Ca, an increase in Hyp, and a 50% decrease in Ca:Hyp. Fluorescent labels given after 8 days of treatment were either diffuse or absent in calcified sections from rats given 125 ng, indicating impaired mineralization. The 25 and 125 ng doses produced hypercalcemia with normal serum phosphate. There was a dose-related increase in serum immunoreactive bone gla protein (BGP) and serum 1,25(OH)2D3 and a decrease in serum 25 (OH)D3. At the 5 ng dose, no adverse effects were seen on body growth. With 25 ng and 125 ng, growth was inhibited. Increased serum urea nitrogen and histologic evidence of nephrocalcinosis occurred at the 125 ng dose. When 125 ng was given for 12 days and then withdrawn for 6 days, systemic toxicity decreased and bone Hyp and Ca increased so that Ca:Hyp remained low and comparable to that of rats treated with 1,25(OH)2D3 continuously We conclude that pharmacologic doses of 1,25(OH)2D3 stimulate trabecular bone matrix formation but produce impairment of mineralization, despite a high Ca×Pi product.

Human parathyroid hormone (1–34) and salmon calcitonin do not reverse impaired mineralization produced by high doses of 1,25 dihydroxyvitamin D3

SummaryWe have reported recently that pharmacologic doses of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulated bone matrix formation but impaired mineralization. The objective of this study was to determine if parathyroid hormone (hPTH 1-34) or calcitonin (sCT) would mineralize the osteoid induced by 1,25(OH)2D3 in rat long bones. In one experiment, male Sprague-Dawley rats were given daily subcutaneous injections of vehicle: 8 μg hPTH(1-34); 125 ng 1,25(OH)2D3; or both 8 μg hPTH and 125 ng 1,25(OH)2D3 per 100 g body weight for 12 days. In a second experiment, rats received daily injections of vehicle: 2 U sCT; 125 ng 1,25(OH)2D3; or both 2 U sCT and 125 ng 1,25(OH)2D3 per 100 g body weight for 18 days. Calcium (Ca), hydroxyproline (Hyp), and dry weight (DW) of the distal femur and serum calcium, phosphate, and serum bone Gla protein (BGP) were measured. In rats given both 1,25(OH)2D3 and hPTH, total bone DW and Hyp increased (P<.01) without a corresponding increase in bone Ca so that Ca/Hyp decreased 47% (P<.01) from control and remained comparable to values for rats treated with 1,25(OH)2D3 alone. In rats treated with both 1,25(OH)2D3 and sCT, total bone DW and Hyp increased while Ca decreased so that Ca/Hyp decreased 38% from control (P<.05), and remained comparable to values for rats treated with 1,25(OH)2D3 alone. These results indicate that hPTH or sCT, given by intermittent injection to rats for 12 or 18 days respectively, failed to mineralize the osteoid induced by high doses of 1,25(OH)2D3.