The effects of 24R,25-dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum.

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.     

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.     

A specific binding protein for 1 alpha,25-dihydroxyvitamin D in the chick embryo chorioallantoic membrane

A 3.7 S binding protein for the steroid hormone and vitamin D metabolite 1 alpha-25-dihydroxyvitamin D (1,25-(OH)2-D) was observed in high salt cytosol extracts of chick embryo chorioallantoic membrane. The binding protein was characterized after partial purification of cytosol extracts by ammonium sulfate fractionation. The binding of 1,25-(OH)2-D was saturable, had a high affinity (Kd = 0.16 nM), and was specific for hormonally active vitamin D metabolites. Analysis of the displacement of [3H]1,25-(OH)2-D by unlabeled analogues showed the affinities of vitamin D metabolites to be in the order of 1,25-(OH)2-D = 1,24R,25-(OH)3-D much greater than 25-OH-D = 1-OH-D greater than 24R,25-(OH)2-D. Hormone binding was sensitive to pretreatment with sulfhydryl-blocking reagents. The chorioallantoic membrane 1,25-(OH)2-D-binding protein associated with the chromatin fraction after homogenization of membranes in low salt buffer, and bound to DNA-cellulose columns, eluting as a single peak at 0.215 M KCl. These findings support identification of this 1,25-(OH)2-D-binding protein as a steroid hormone receptor, with properties indistinguishable from 1,25-(OH)2-D receptors in other chick tissues. The chorioallantoic membrane functions in the last third of embryonic development to reabsorb calcium from the eff shell for deposition in embryonic bone. 1,25-(OH)2-D binding activity in the chorioallantoic membrane increased 4- to 5-fold from day 12 to day 16 of incubation, immediately preceding the onset of shell reabsorption. This finding suggests that 1,25-(OH)2-D may act to regulate shell mobilization and transepithelial calcium transport by the chorioallantoic membrane. Finally, the similarity of shell mobilization to bone resorption, which is also stimulated by 1,25-(OH)2-D, suggests that the chorioallantoic membrane is a useful alternate model for the study of 1,25-(OH)2-D action on bone mineral metabolism.     

A high-affinity cytosol binding protein for 1 alpha,25-dihydroxycholecalciferol in the uterus of Japanese quail.

Cytosol fractions prepared from the uterine mucosa of egg-laying Japanese Quail were analysed for binding of the metabolites of cholecalciferol. When the uterus was incubated at 37 degrees C with various radioactive metabolites of cholecalciferol, the nuclear fraction incorporated only 1 alpha,25-dihydroxy[3H]cholecalciferol. When the uterus was incubated at 0 degree C with 1 alpha,25-dihydroxy[3H]cholecalciferol, most of the radioactivity was found in the cytosol. Translocation of 1 alpha,25-dihydroxy[3H]cholecalciferol from the cytosol to the nucleus was temperature-dependent. The addition of 100-fold excess amounts of unlabelled 1 alpha-25-dihydroxycholecalciferol significantly diminished the nuclear binding of 1 alpha,25-dihydroxy[3H]cholecalciferol. The cytosol fraction contained a 3.5 S macromolecule that specifically binds 1 alpha,25-dihydroxy[3H]cholecalciferol. The dissociation constant was 0.39 nM and the maximal binding was 55 fmol/mg of protein. These results strongly suggest that the uterus in egg-laying birds is a target organ or 1 alpha,25-dihydroxycholecalciferol.     

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

Functional involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane.

Carbonic anhydrase activity was demonstrated in the chick-embryonic chorioallantoic membrane and was correlated with the Ca2+-transport activity of the membrane. It is inhibited by sulphonamides and is expressed in the chorioallantoic membrane in an age-dependent fashion during embryonic development. Ca2+ uptake by the chorioallantoic membrane in vivo also increases in a similar age-dependent manner. The temporal increase in these activities is coincident with calcium deposition in the embryonic skeleton. Incubation of the chorioallantoic membrane in ovo with sulphonamides specifically inhibits both the carbonic anhydrase and the Ca2+ uptake activities of the membrane in vivo. Enzyme histochemistry revealed the carbonic anhydrase activity is localized in the Ca2+-transporting ectodermal cells of the chorioallantoic membrane. These results, taken together, indicate that carbonic anhydrase may be functionally important in the Ca2+-transport activity of the chorioallantoic membrane.     

The appearance of 1,25-dihydroxyvitamin D3 receptor during chick embryo development

The appearance of the 1,25-dihydroxyvitamin D3 receptor in intestine, kidney, and chorioallantoic membrane of chick embryo was followed by sucrose density gradient sedimentation analysis and Scatchard plot analysis. The receptor from each of these organs sediments as a single 3.7S component. At 19 days of embryonic life, intestine had the highest specific 1,25-dihydroxyvitamin D3 binding activity followed by kidney and chorioallantoic membrane. The 1,25-dihydroxyvitamin D3 binding activity increased gradually at 12-15 days and rapidly until 20 days in intestine. In kidney, this protein increased rapidly from 12 to 16 days and did not change subsequently. In chorioallantoic membrane, the receptor increased slowly from 8 through 15 days, rapidly until 19 days, and decreased at 20 days. The injection of hydrocortisone into the chick embryo at 10 days increased receptor number in intestine, kidney, and chorioallantoic membrane by a factor of 2 at 12 days. Injection of this hormone after this time had little or no effect.     

Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

Structural analogs of 1alpha,25-dihydroxycholecalciferol: preparation and biological assay of 1alpha-hydroxypregnacalciferol.

The synthesis of 1alpha-hydroxypregnacalciferol, a side chain analog of 1alpha,25-dihydroxycholecalciferol (1alpha,25-dihydroxyvitamin D3), is described. Pregnenolone acetate was converted in five steps to 5-pregnen-1alpha,3beta-diol. Conversion of the diol to pregna-5,7-diene-1alpha,3beta diol diacetate followed by ultraviolet irradiation gave the corresponding previtamin derivative. Thermal isomerization, hydrolysis and chromatography then furnished the desired analog, 1alpha-hydroxypregnacalciferol. The compound was tested in vivo for its effect on intestinal calcium transport, serum calcium and phosphate levels and bone calcification, and in vitro for its effect on bone resorption. When given to intact rats, either as a single dose or in repeated daily doses, the analog even at high dose levels, exhibited no biological activity. The compound stimulated bone resorption in vitro, but only at high concentrations.     

Co-expression of the proto-oncogene fos (c-fos) and an embryonic interferon (ovine trophoblastin) by sheep conceptuses during implantation.

Expression of the c-fos proto-oncogene by ovine conceptuses was analyzed by Northern and slot blots and indirect immunohistofluorescence in relation to the expression of the embryonic interferon-alpha (oTP) during implantation. c-fos was expressed initially in the trophoblast, and then in the allantois, when this tissue began to develop (day 17). In the embryonic tissues, the c-fos proto-oncogene was weakly expressed up to day 22 and increased thereafter. In the trophoblast, the expression of c-fos proto-oncogene was transient, occurring when the oTP gene was transcribed at a maximal level at the beginning of implantation (days 14-15), and decreased thereafter, following the pattern of oTP gene expression. This decline is due essentially to the arrest of c-fos and oTP gene expression by the trophoblastic cells which established cellular contacts with the uterine epithelium during the implantation process.     

Identification, emergence and mobilization of circulating endothelial cells or progenitors in the embryo

Using quail-chick parabiosis and QH1 monoclonal antibody analysis, we have identified circulating endothelial cells and/or progenitors in the embryo. These cells were already present early in ontogeny, before the third embryonic day. Under normal conditions, they integrated into most tissues but remained scarce. When experimental angiogenic responses were induced by wounding or grafts onto the chorioallantoic membrane, circulating endothelial cells were rapidly mobilized and selectively integrated sites of neoangiogenesis. Their mobilization was not dependent on the presence of the bone marrow as it was effective before its differentiation. Surprisingly, mobilization was not effective during sprouting angiogenesis following VEGF treatment of chorioallantoic membrane. Thus, embryonic circulating endothelial cells were efficiently mobilized during the establishment of an initial vascular supply to ischemic tissues following wounding or grafting, but were not involved during classical sprouting angiogenesis.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Differential effects of Vitamin D analogs on bone formation and resorption

Deficiency in Vitamin D and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization, suggesting that Vitamin D analogs might stimulate osteoblastic bone formation and mineralization. In this study, we compare the effect of selected Vitamin D analogs and active metabolite, 1alpha,25-dihydroxyvitamin D(3), 19-nor-1alpha, 25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) or 1alpha,25-dihydroxyvitamin D(2) on bone formation and resorption. In a mouse calvariae bone primary organ culture system, all Vitamin D analogs and metabolite tested-stimulated collagen synthesis in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was the most efficacious among three. 19-nor-1alpha, 25-dihydroxyvitamin D(2) and 1alpha,25-dihydroxyvitamin D(2) showed similar potencies and 1alpha,25-dihydroxyvitamin D(3) was less potent than others. Osteocalcin was also up-regulated in a dose-dependent manner, suggesting that the three Vitamin D analogs have the equal potencies on bone formation. 25-Hydroxyvitamin D-24-hydroxylase expression was induced in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was less potent than other two compounds. In a mouse calvariae organ culture, all induced a net calcium release from calvariae in a dose-dependent manner, but the potency is in the order of 1alpha,25-dihydroxyvitamin D(2) congruent with1alpha,25-dihydroxyvitamin D(3)>19-nor-1alpha, 25-dihydroxyvitamin D(2). In a Vitamin D/calcium-restricted rat model, all caused an elevation in serum calcium in a dose-dependent manner. There is no significant difference between 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) in potencies, but 19-nor-1alpha, 25-dihydroxyvitamin D(2) is at least 10-fold less potent than the other two compounds. Our results suggest that Vitamin D analogs have direct effects on bone resorption and formation, and 19-nor-1alpha, 25-dihydroxyvitamin D(2) may be more effective than 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) on stimulating anabolic bone formation.