The effects of vitamin D metabolites on bone collagen synthesis were assessed in organ cultures of fetal rat calvaria by measuring the incorporation of a 2-h pulse of [ :) H]proline into collagenase-digestible (CDP) and non-collagen protein (NCP) by using purified bacterial collagenase. Addition of la,25-dihydroxyvitamin Da (la,25(OH)2D.i) to cultures containing 5% vitamin D-deficient serum produced a dose-related inhibition of incorporation of proline into CDP over the range of 10"" to 10~7 M at 24 h. Similar inhibition was obtained with la,25(OH)2D,) in 4-day cultures by using medium supplemented with bovine serum albumin instead of serum. The effect on the labeling of CDP was selective and in most experiments there was no effect on incorporation of proline into NCP. la,24R,25-trihydroxyvitamin D. t (la,24R,25(OH).,D.,) at K)-9 -10-7 M also inhibited the labeling of CDP, but three other compounds, 25-hydroxy-, 24R,25-dihydroxy-, and 24S,25-dihydroxyvitamin D.i, had no consistent effect. To test whether a metabolite of vitamin D in serum might affect collagen synthesis, bones were cultured with 1-10% serum from vitamin D-deficient and vitamin D-treated animals. There was no greater labeling of CDP and NCP with vitamin D-sufficient serum. We conclude that the vitamin D metabolites which are most active in stimulating bone resorption, la,25(OH) 2 D:t and la,24R,25(OH). } D.), also inhibit bone collagen synthesis in vitro. No evidence for a stimulatory effect on bone collagen synthesis was obtained by using the currently available natural metabolites of vitamin D or by comparing serum from vitamin D-deficient or -treated animals. (Endocrinology 102: 731 , 1978) V ITAMIN D appears to be required for skeletal growth and mineralization. Hence, it seems paradoxical that the active form of the vitamin, la,25-dihydroxyvitamin D3 (la,25(OH) 2 D;3), is a potent stimulator of bone resorption (1). One explanation for this apparent discrepancy is that intestinal absorption of calcium and phosphate is increased to a greater extent by the active metabolites of vitamin D than is bone resorption. This would increase the serum calcium and phosphate concentrations, thus enhancing bone mineralization, while at the same time suppressing parathyroid hormone (PTH) so that bone resorption might not be increased in vivo. It may also be important to stimulate bone resorption in order to maintain skeletal growth because, at least in bone remodeling, new bone formation generally occurs at resorption sites. In either case, vitamin D would have a permissive, rather than a direct, role in bone growth. The initial stimulus to bone formation might be from other growth-regulating hormones such as insulin and somatomedin(s) (2, 3), by local feedback effects of changes in mineralization, or by serum calcium and phosphate concentration themselves (4).An attractive alternative is that vitamin D or its metabolites have a direct stimulatory effect, either upon collagen synthesis by osteoblasts or upon bone mineralization. In vivo experiments (5) and...

Parathyroid hormone decreased the incorpo- (5), and net accumulation of bone collagen decreases (6). Similarly, PTH inhibits the synthesis of collagen in osteoblast-like cells derived from 2-to 3-day mouse calvaria by sequential enzymatic digestion (7). The molecular mechanism by which PTH alters collagen synthesis in these bone cultures is unknown. Because procollagen mRNA levels appears to be the primary regulatory site for changes in collagen synthesis in embryonic chicken calvaria (8), a likely hypothesis is that PTH regulates collagen synthesis in fetal rat bone by altering the level of procollagen mRNA. On the other hand, PTH is a potent stimulator of bone resorption (9, 10) and could have a posttranslational effect on collagen levels by enhancing the degradation of newly synthesized collagen.One objective of the present study was to determine whether the ability of PTH to inhibit collagen synthesis in cultures of fetal rat calvaria could be correlated with alterations in the level of functional procollagen mRNA. Another was to assess the effect of PTH on the release of newly synthesized collagen from these calvaria into the culture medium. Our results demonstrate that PTH decreases both collagen synthesis and the bone level of procollagen mRNA but has only a small effect on the release of collagen into the medium, indicating that PTH regulates collagen synthesis primarily through an effect on procollagen mRNA levels.MATERIALS AND METHODS Culture Method. The frontal and parietal bones were removed from 21-day fetal rats (Sprague-Dawley; Charles River Breeding Laboratories) and split along the sagittal suture. Half calvaria were cultured at 370C under a humidified atmosphere of 5% C02/95% air in 25-ml erlenmeyer flasks containing 2 ml of modified BGJ medium (11) supplemented with bovine serum albumin at 1 mg/ml or with 5% human serum that had been heated at 56°C for 30 min. The concentration of unlabeled proline in the medium was 1 mM except where indicated. Half calvaria were cultured for 24 hr with continuous shaking (60 oscillations per min) at 370C. Either natural bovine PTH 1-84 (National Institutes of Health) or the synthetic bovine 1-34 peptide (Beckman) was added to cultures in a solution containing 1 mg of bovine serum albumin per ml. During the final portion of the culture period, 5-10,uCi of [2,3-3H]proline (30 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels; New England Nuclear) was added to each flask for 15-120 min. To terminate the culture each calvarium was removed, extracted with 5% trichloroacetic acid, acetone, and ether and then dried, weighed, and homogenized in 1.0 ml of 0.5 M acetic acid.The incorporation of [3H]proline into CDP and NCP was determined by using repurified bacterial collagenase (Worthington) according to the method of Peterkofsky and Diegelmann (12) and was linear for at least 120 min in both control and PTH-treated calvaria (Fig. 1). The percentage collagen synthesized by the bones was corrected for the relative abundance of proline residues in collagen compared to NCP ...

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The mechanism of congenital osteopetrosis in microphthalmic (mi) mice has been examined in bone organ cultures. Resorption was measured by the release of previously incorporated 45Ca in fetal long bones and newborn calvaria from mi mice and heterozygous or homozygous normal litter mates. Bones from mi mice showed a generalized resorption defect with decreased spontaneous or control resorption and failure to respond to parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxy vitamin D3, vitamin A, or osteoclast activating factor (OAF) from human peripheral leukocytes or mouse spleen cells. Bones from heterozygotes showed a smaller response to PTH than bones from homozygous normals. Mutant bones failed to show an increase in lysosomal enzyme release in response to PTH or vitamin A, agents which increased release from bones of homozygous normals. Proline incorporation into collagenase-digestible protein was similar in cultures of normal and mutant bone and was inhibited by PTH and OAF. These results indicate that congenital osteopetrosis in mi mice is due to a generalized defect in the function and hormonal response of osteoclasts and suggests that this cell line is separate from the osteoblast cell line which shows no impairment of hormonal response.

An analog of bobine PTH [nle-8, nle-18, tyr-34 bPTH-(1-34) amide, (PTH-Ana)] which is a potent stimulator of renal adenylate cyclase has been compared with the native hormone bPTH-(1-84) and the biologically active amino terminal portion, bPTH-(1-34), for its effects on bone resorption and bone collagen synthesis in organ culture. All three compounds stimulated the release of previously incorporated 45Ca from cultured fetal rat long bone shafts with similar dose-response curves at 10(-9) to 3 X 10(-8) M. All three compounds inhibited bone collagen synthesis as measured by incorporation of proline into collagenase digestible protein, whereas incorporation into noncollagen protein was not inhibited. The effects were dose related and decreases in percent collagen synthesis were significant at 10(-9) M. Thus PTH-Ana appears to have the same effects on bone resorption and collagen synthesis as bPTH-(1-84) and (1-34) and is likely to be a valid probe for investigating PTH receptors in bone as well as in kidney.