No abstract

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-␤, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100°C and separable from the bulk of the remaining alcohol-and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a Mr of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.intracrine ͉ selective estrogen receptor modulator ͉ steroid S ex steroids cause direct genomic transcriptional effects through specific receptors that contain ligand binding, dimerization, DNA binding, and gene transactivation domains. They also cause indirect stimulatory or inhibitory effects through complexes composed of activated hormone receptor and other transcription factors or through variations in the activation potential of other transcriptional components (1-6) The skeleton is a well recognized target for sex steroids. Bone fragility is notable when sex steroid levels fall in women after menopause, in elderly males, or after sex organ ablation (7-11). In such cases, bone loss follows a release from native constraints on bone resorption that largely result from changes in growth regulators expressed by osteoblasts and from opposing effects on osteoblast and osteoclast activation and apoptosis, leading to an overall increase in bone remodeling (12-16). Importantly, imbalances in bone remodeling are restored by sex hormone replacement therapy. Regardless of a clear benefit for bone, use of native sex steroids risks inappropriate gene activation in other tissues. The possibility of breast, cardiovascular, and prostate disease that can occur in this context (17-19) has driven the search for sex hormone receptor modulators with tissue-or function-restricted effects. However, the value of agents like these may be complicated by steroid and precursor metabolizing enzymes expressed by osteoblasts (3,20,21).Many cells u...

The hypophosphatemic (Hyp) mouse is a model for human familial hypophosphatemic rickets. To test the hypothesis that there is an osteoblastic defect in these animals, serum osteocalcin levels were measured in Hyp mice and their normal littermates. Furthermore, the effects of phosphorus deprivation, phosphorus loading, and 1,25-dihydroxyvitamin D3 administration on serum osteocalcin levels were examined. Osteocalcin was purified from mouse hindlimbs, and a polyclonal antibody to this material was produced in a goat. The antibody recognized native and decarboxylated mouse osteocalcin, but could not recognize osteocalcin from several other species. A RIA was developed which had a minimal detection limit of 0.4 nmol/liter (2.2 micrograms/liter) and half-maximal displacement at 2.7-3.3 nmol/liter (14.8-18.2 micrograms/liter). The intraassay coefficient of variation was 6.4%, while the interassay coefficient of variation was 12%. Dilutions of mouse serum samples varied by less than 15%. Analytical recovery was typically greater than 90%. Serum osteocalcin concentrations in Hyp and normal mice were shown to decrease with age. However, circulating osteocalcin levels in Hyp mice were higher than those in their normal littermates regardless of the age of the animal (P less than 0.001). One week of a high phosphorus diet resulted in an increase in serum phosphate in normal and Hyp mice, but serum osteocalcin concentrations were unaffected. On the other hand, dietary phosphorus deprivation for 4 weeks resulted in comparable hypophosphatemia in both Hyp and normal mice, and serum osteocalcin increased in both groups of animals. Intraperitoneal injection of 30 ng/day 1,25-dihydroxyvitamin D3 for 7 days resulted in a 215 +/- 33% increase in serum osteocalcin in normal animals, while the same regimen produced a 250 +/- 29% decrease in the Hyp mouse. Our results are consistent with the hypothesis that abnormal osteoblastic activity is present in Hyp mice. Furthermore, hypophosphatemia may be a general regulator of osteocalcin synthesis or secretion in the mouse.

Osteocalcin is initially synthesized as an 11 kD molecule consisting of a 23-residue translocation signal peptide that is cleaved during translation, a 26-residue propeptide that targets the protein for gamma-carboxylation, and the 49-residue mature protein. Although the majority of newly synthesized osteocalcin is deposited into bone matrix, a small amount can be detected in blood, and it is this characteristic that has led to its current clinical use as a specific index of osteoblastic activity. Nothing is known, however, about the fate of the propeptide. If osteocalcin and the propeptide are cosecreted, then the concentration of the propeptide could also be useful as a marker of osteoblastic function and, further, may be superior to osteocalcin because it would be unaffected by binding to bone. To test this hypothesis, we synthesized a peptide corresponding to 21 residues of the osteocalcin propeptide from humans and produced a polyclonal antibody to this peptide. Human sera were screened for the presence of the propeptide, and the human osteosarcoma cell line MG-63 was tested for secretion of the propeptide. We could not detect any osteocalcin propeptide in sera from normal adults or individuals with renal failure or primary hyperparathyroidism or those on long-term coumadin therapy. Likewise there was no propeptide present in media from cells grown in the presence of vitamin K, 1,25-(OH)2D3, warfarin, or warfarin plus 1,25-(OH)2D3. In contrast, the cell extract, characterized by high-performance liquid chromatography, contained mature osteocalcin, free propeptide, and the proosteocalcin precursor when cells were grown in the presence of 1,25-(OH)2D3 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

We compared the effects of parathyroid hormone (PTH1-34) and parathyroid hormone-related protein (PTHrp1-34) on osteocalcin release in the isolated rat hindlimb and in intact and thyroparathyroidectomized (TPTX) rats. PTH1-34 suppressed osteocalcin release from perfused rat hindquarters, while PTHrp1-34 had no effect on osteocalcin release, despite comparable stimulation of cAMP production. Similarly, serum osteocalcin declined in the intact and TPTX animals by 5 h after a single dose of PTH1-34, while there was no response to PTHrp1-34. Chronic administration of PTH1-34 or PTHrp1-34 produced comparable hypercalcemia and hypophosphatemia in sham-operated and TPTX animals. Chronic PTH1-34 administration produced significant increases in serum osteocalcin both in the sham-operated rats and in the TPTX animals. However, in animals chronically treated with PTHrp1-34, there was no change at any time point in osteocalcin in either sham-operated or TPTX rats. These differences could be inherent or merely due to potency differences between the two peptides.