Different Effects of Insulin and Insulin-Like Growth Factors I and II on Osteoprogenitors and Adipocyte Progenitors in Fetal Rat Bone Cell Populations

We investigated the effects of insulin (1–1,000 nM), insulin-like growth factor (IGF)-I, and IGF-II (3–100 nM each) alone or together with 10 nM dexamethasone (DEX) or 10 nM 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) on proliferation and differentiation of adipocyte and osteoblast progenitors in bone cell populations derived from fetal rat calvaria. The effects on differentiation were evaluated by counting the number of bone or osteoid nodules and adipocyte colonies and the effects on proliferation, by measuring their size by image analysis. The types of cells studied were 1,25(OH)₂D₃- and DEX-responsive adipocyte progenitors and DEX-dependent and independent osteoprogenitors. Both IGF-I and IGF-II stimulated osteoprogenitor differentiation both alone and in the presence of DEX, while insulin stimulated osteoprogenitor differentiation only in the absence of DEX. Neither IGF-I/-II nor insulin affected proliferation of osteoprogenitors. Insulin had little effect on adipocyte differentiation by itself but strongly stimulated differentiation in the presence of either 1,25(OH)₂D₃ or DEX, while IGF-II stimulated adipocyte differentiation in both the absence and presence of 1,25(OH)₂D₃ or DEX. IGF-I by itself or in the presence of DEX strongly stimulated adipocyte cell differentiation but had little effect in the presence of 1,25(OH)₂D₃. Our results demonstrate that insulin, IGF-II, and IGF-I have specific and different effects on the differentiation and proliferation of different groups of progenitor cells.     

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Summary Vitamin D₃ metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)₂D₃ whereas resting zone chondrocytes respond primarily to 24,25(OH)₂D₃. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)₂D₃, and 24,25(OH)₂D₃ on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influex was measured as incorporation of⁴⁵Ca. Efflux was measured as release of⁴⁵Ca from prelabeled cultures into fresh media. The pattern of⁴⁵Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of⁴⁵Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10⁻⁸–10⁻⁹ M 1,25(OH)₂D₃ to growth zone chondrocyte cultures results in a dose-dependent increase in⁴⁵Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10⁻⁶–10⁻⁷ M 24,25(OH)₂D₃ to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes.⁴⁵Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.     

Suppression of growth plate chondrocyte proliferation by corticosteroids

Growth depression is a side effect of high-dose glucocorticoid therapy in childhood. It is partially mediated by alterations of the somatotropic hormone axis and partially by direct local effects on growth plate chondrocytes. The mechanisms of interaction of corticosteroids and somatotropic and calciotropic hormones at the cellular level were recently investigated in more detail, using experimental models of primary cultures of growth plate chondrocytes. In proliferative chondrocytes, growth hormone (GH) and the calciotropic hormones parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1α,25(OH)₂D₃] increase cell proliferation via stimulation of paracrine insulin-like growth factor-I (IGF-I) secretion. Corticosteroids decreased GH, and PTH or 1α,25(OH)₂D₃ stimulated cell growth in a dose-dependent manner. Corticosteroids in high doses reduced the expression of the GH receptor and type 1 IGF receptor. But the main antiproliferative molecular effect of corticosteroid was the reduction in basal and hormone-stimulated IGF-I secretion. The in vitro results are in accordance with the observation in animal experiments and in children treated with corticosteroids, demonstrating that the growth-depressing effect of corticosteroids can be compensated for by supraphysiological doses of GH or IGF-I. Received: 16 May 1999 / Revised: 31 January 2000 / Accepted: 8 February 2000     

Hormonal regulation of vitamin D receptor levels and osteocalcin synthesis in human osteosarcoma cells

1,25(OH)₂D₃ was found to regulate its own receptor levels via an increase in corresponding mRNA levels in human osteoblast-like osteosarcoma cells (MG-63). In addition, exposure of the cells for 24h to dexamethasone, estradiol, retinoic acid, or triiodothyronine resulted in a dose-dependent accumulation of hVDR mRNA. Combination of 1,25(OH)₂D₃ with any other hormone used in this study did not result in an additive increase in hVDR mRNA levels. Progesterone or dihydrotestosterone did not influence hVDR mRNA levels. Of the studied hormones, only 1,25(OH)₂D₃ was alone able to stimulate the synthesis and secretion of osteocalcin. Compared with 1,25(OH)₂D₃, the combination of 1,25(OH)₂D₃ and retinoic acid resulted an increased synthesis of osteocalcin. In contrast, the combination of 1,25(OH)₂D₃ with dexamethasone, estradiol, or triiodothyronine diminished the stimulatory effect of 1,25(OH)₂D₃. A complex interaction of several different hormone receptors seems to occur within the regulatory regions of hVDR and osteocalcin genes, or at the level of translation, resulting, in each case, a finely adjusted vitamin D receptor and osteocalcin expression.     

Effect of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion

Summary The active vitamin D metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] causes marked suppression of both pre-proparathyroid hormone messenger RNA (pre-proPTH mRNA) and parathyroid hormone (PTH) secretion. These effects are dose dependent and reversible when tested in anin vitro primary tissue culture cell system using normal bovine parathyroid cells. In the current studies, the precursors of 1,25(OH)₂D₃ and the related metabolite 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], were used in the same culture system to test for possible regulatory effects. The results were compared with identically prepared cells exposed to 1,25(OH)₂D₃. In short-term studies (30–120 minutes), none of the vitamin D-related compounds produced any effect on PTH secretion. In long-term studies (24–48 hours, using primary tissue culture in the presence of test agents), neither vitamin D₃ nor 25(OH)D₃ affected PTH secretion or pre-proPTH mRNA over the concentration range 10⁻¹¹–10⁻⁷M. On the other hand, 24,25(OH)₂D₃ produced significant suppression of both pre-proPTH mRNA (77% of control,P<.01) and PTH secretion (75% of control,P<.005) at 10⁻⁷ M. By comparison, 10⁻¹¹ M 1,25(OH)₂D₃ produced levels of suppression (25–30%) of both pre-proPTH mRNA and PTH secretion comparable to 10⁻⁷ M 24,25(OH)₂D₃, while even greater suppression (40–50%) occurred at 10⁻⁹-10⁻⁷ M 1,25(OH)₂D₃. From these studies, we conclude that vitamin D₃ and 25(OH)D₃ do not have significant effects on PTH synthesis and secretion over the range of doses tested. Compared with 1,25(OH)₂D₃, 24,25(OH)₂D₃ exhibits mild suppression at pharmacologic concentrations. The effect of 24,25(OH)₂D₃ prabably occurs through weak interaction of 24,25(OH)₂D₃ with the 1,25(OH)₂D₃ receptor.     

Modulation by epidermal growth factor of the basal 1,25(OH)2D3 receptor level and the heterologous up-regulation of the 1,25(OH)2D3 receptor in clonal osteoblast-like cells

Summary The effects of epidermal growth factor (EGF) on basal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) receptor level and on parathyroid hormone (PTH)-induced 1,25-(OH)₂D₃ (OH)₂D₃ receptor up-regulation were studied in the phenotypically osteoblastic cell line UMR 106. EGF in concentrations exceeding 0.1 ng/ml reduced the number of 1,25(OH)₂D₃ binding sites without changing the binding affinity. Maximal reduction was 30% at about 1 ng/ml. This reduction was independent of a change in cAMP content. EGF dose-dependently attenuated both PTH-induced 1,25(OH)₂D₃ receptor up-regulation and PTH-stimulated cAMP production without and effect on the ED₅₀ of the PTH effects. For both PTH responses the IC₅₀ and the maximal effective dose were similar, 0.1 ng/ml an 1 ng/ml EGF, respectively. Reduction was first seen at 0.01 ng/ml EGF. At this concentration. EGF reduced PTH-stimulated 1,25-(OH)₂D₃ receptor binding without an inhibition of the cAMP response. Time-course studies with 1 ng/ml EGF revealed that at 2 h preincubation EGF reduced the heterologous up regulation by PTH, and maximal inhibition was seen after 4 h. In contrast, PTH-stimulated cAMP production was just significantly inhibited only after 6 h, with 60% inhibition after 24 h preincubation. The effects of prostaglandin E₂ and forskolin on both 1,25(OH)₂D₃ binding and cAMP production were inhibited in a similar fashion. On the other hand, dibutyryl cAMP- and 3-isobutyl-1-methylxanthinestimulated 1,25(OH)₂D₃ binding were not affected by EGF. Taken together, our results demonstrate that EGF reduces both the basal number of 1,25(OH)₂D₃ binding sites and the heterologous up-regulation of the 1,25(OH)₂D₃ receptor. The current data suggest that EGF reduces heterologous upregulation of the 1,25(OH)₂D₃ receptor independent of as well as dependent on the cAMP messenger system. The EGF effect is not primarily located at the PTH receptor, at cAMP phosphodiesterase, or at protein kinase A level.     

Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2)

Summary Rat osteogenic sarcoma cells have been used widely as a model system to study actions of 1,25(OH)₂D₃ and other hormones in osteoblastlike cells. However, some of the pleiotypic manifestations of hormones in these cells vary greatly dependent upon the cell population density and other conditions of culture. Therefore, we have studied the effect of cell density on the relationship between 1,25(OH)₂D₃ and the initial⁴⁵Ca accumulation in ROS 17/2 cells in order to establish conditions suitable for studying the effect of 1,25(OH)₂D₃ on calcium fluxes in these cells. Cells were grown in the presence and absence of 1,25(OH)₂D₃ for 48 hours and then incubated for 4 min in the culture medium containing 0.5 μCi/ml of⁴⁵CaCl₂. In high population density cultures, 0.25–1.0 pg/ml of 1,25(OH)₂D₃ stimulated the intracellular accumulation of⁴⁵Ca (cpm/mg protein), whereas 80 pg/ml or higher concentrations inhibited accumulation of⁴⁵Ca. In low density cultures, concentrations less than 80 pg/ml had no effect, 80–120 pg/ml increased the intracellular accumulation, and as much as 200 pg/ml failed to show the inhibitory effect. These results indicate that the ROS 17/2 cell responses to 1,25(OH)₂D₃ are biphasic—low concentrations stimulating and high concentrations inhibiting⁴⁵Ca accumulation. The sensitivity of the cells to 1,25(OH)₂D₃ increases as the cell population density increases. These observations suggest that the culture density and dose-response relationship must be carefully defined inin vitro studies utilizing osteogenic cell culture systems.     

Effect of 24R,25-dihydroxyvitamin D3 on the formation and function of osteoclastic cells

Summary Previous reports demonstrated that the administration of large doses of 24R,25-dihydroxyvitamin D₃ [24R,25(OH)₂D₃] to animals with normal vitamin D supply causes an increase in bone volume with reduced bone resorption and decreased osteoclast number. The present study was undertaken to clarify if 24R,25(OH)₂D₃ has any inhibitory effect on the formation and function of osteoclasts. The effect of 24R,25(OH)₂D₃ on the formation of osteoclastic cells was examined by measuring the number of tartrate-resistant acid phosphatase-positive multinucleated cells (MNCs) formed from hemopoietic progenitor cells obtained from spleens of 5-fluorouracil-treated mice. Treatment with 1,25(OH)₂D₃ or parathyroid hormone fragment 1–34 [PTH(1–34)] stimulated osteoclast-like MNC formation in a dose-dependent manner. Addition of 24R,25(OH)₂D₃ alone showed a weak stimulatory effect on MNC formation at 10^-6 M, which appeared to be due to its binding to 1,25(OH)₂D₃ receptors. In contrast, when 24R,25(OH)₂D₃ was added together with 1,25(OH)₂D₃ or PTH(1–34), it inhibited osteoclast-like MNC formation stimulated by these hormones. A significant inhibition of MNC formation was observed with 10^-7M 24R,25(OH)₂D₃, and the stimulatory effect of 1,25(OH)₂D₃ or PTH(1–34) was almost completely eliminated with 10^-6 M 24R,25(OH)₂D₃. Neither 24S,25(OH)₂D₃ nor 25(OH)D₃ exhibited a similar inhibitory effect. The effect of 24R,25(OH)₂D₃ on the resorptive function of osteoclasts was examined by measuring the formation of resorption pits by mouse bone cells on dentine slices. Treatment with 24R,25(OH)₂D₃ also inhibited the resorption pit formation stimulated by 1,25(OH)₂D₃ or PTH(1–34) with similar dose response. These results demonstrate that 24R,25(OH)₂D₃ has a specific inhibitory effect on the formation and function of osteoclastic cells stimulated by 1,25(OH)₂D₃ or PTH, and suggest that these effects of 24R,25(OH)₂D₃ may play role in the regulation of bone metabolism by modulating the actions of osteotropic hormones on osteoclastic bone resorption.     

Effects of vitamin D-binding protein on bone resorption stimulated by 1,25 dihydroxyvitamin D3

Summary Vitamin D and its metabolites are tightly bound to the serum vitamin D-binding protein (DBP) and only the free hormone is considered to be physiologically active. On the other hand, DBP could interact with cell membranes and even favor its intracellular entry. The present study was undertaken to examine the effects of DBP on bone resorption stimulated by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Forelimb bones from 19-day-old fetal rats were cultured for 5 days in the presence of purified human or rat serum albumin (hSAP or rSAP) and 1,25(OH)₂D₃, with or without human or rat DBP (hDBP or rDBP). Bone resorption was assessed by measuring the release of previously incorporated⁴⁵Ca. We found that the resorptive response to 1,25(OH)₂D₃ was minimally altered by hDBP (5 μM). The minimal effects of hDBP on 1,25(OH)₂D₃ activity on rat bones might be explained by a 6-fold lower affinity of hDBP (1.1×10⁷ M⁻¹) than rDBP (5.9×10⁷ M⁻¹) for 1,25(OH)₂D₃ or by species differences in cellular recognition of DBP. In a homologous rat system, however, rDBP at low (0.5 μM) or physiological (5 μM) concentration significantly decreased 1,25(OH)₂D₃-induced bone resorption. These data therefore support the hypothesis that free rather than DBP-bound 1,25(OH)₂D₃ is physiologically important.     

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

Summary We 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.     

Induction of matrix gla protein synthesis during prolonged 1,25-Dihydroxyvitamin D3 treatment of osteosarcoma cells

Summary The synthesis of matrix Gla protein (MGP) and bone Gla protein (BGP) have been shown to be mutually exclusive in all osteosarcoma cell lines investigated. In the cell lines that produce the respective proteins, synthesis is stimulated by 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) within the first several hours of hormone treatment. In the present studies we have investigated the effects of longer-term treatment with 1,25(OH)₂D₃ in the ROS 17/2 cell line, a cell line that synthesizes BGP constitutively but does not synthesize MGP. In agreement with earlier studies, the rate of BGP synthesis increases within 8 hours of hormone treatment, is maximal by 24 hours, and remains at the maximal rate through 48 hours of 1,25(OH)₂D₃ treatment. The present study is the first to report that the rate of BGP secretion at times beyond 48 hours declines to that of control cultures despite the continued administration of 1,25(OH)₂D₃, and that MGP synthesis is induced in ROS 17/2 cells by 48 hours of 1,25(OH)₂D₃ treatment. At this time, MGP mRNA could be detected by northern blot analysis and MGP secretion could be demonstrated by radioimmunoassay of culture medium. Both the level of MGP message per unit total RNA and the rate of MGP secretion into culture medium increased steadily between 2 and 6 days of 1,25(OH)₂D₃ treatment. The MGP synthesized by the 1,25(OH)₂D₃-treated ROS 17/2 cells was identical to that found in bone by northern blot analysis of message and by western blot analysis of the media antigen. Halfmaximal induction of MGP synthesis was obtained with 0.3 nM 1,25(OH)₂D₃, a 60-fold higher dosage than was required for the half maximal stimulation of BGP synthesis in these cells. Treatment of ROS 17/2 cells with 24,24-F₂1,25(OH)₂D₃ suggests that the observed difference in dose dependence is not due to an increased rate of hormone catabolism.     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

Glucocorticoids increase the 1,25(OH)2D3 receptor concentration in rat osteogenic sarcoma cells

Summary We have used cultured osteoblastlike rat osteogenic sarcoma cells (ROS 17/2) which have receptors for 1,25(OH)₂D₃ and for glucocorticoids, and have examined the modulation of the 1,25(OH)₂D₃ receptor by the potent glucocorticoid triamcinolone acetonide. We report that triamcinolone acetonide caused an increase of the 1,25(OH)₂D₃ receptor concentration in these cells but it did not affect the affinity of the receptor to 1,25(OH)₂D₃; this phenomenon occurred in a dosedependent fashion for triamcinolone (10⁻⁹ to 10⁻⁷ M) with a maximum increase of 1,25(OH)₂D₃ receptor concentration of ⋍twofold. During the culture period, the 1,25(OH)₂D₃ receptor concentration was altered both in untreated as well as in triamcinolone-treated cells, being highest at the early logarithmic phase and diminished progressively as cells approached confluence. However, throughout the culture period, the 1,25(OH)₂D₃ receptor concentration was higher in the triamcinolone-treated cells.     

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

Studies on the mode of action of vitamin D XXXVII 1α-hydroxyvitamin D3: A long-acting 1,25-dihydroxyvitamin D3 analog

Summary This study was undertaken to determine whether 1α-hydroxyvitamin D₃ [1α(OH)D₃] administration to chicks in vivo results in 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] intestinal receptor occupancy and to compare the temporal characteristics of the physiological effects of 1α(OH)D₃ and 1,25(OH)₂D₃ for several days after a single dose of either steroid. Occupied 1,25(OH)₂D₃ receptors of the chick duodenal mucosa were measured by the recently developed exchange assay procedure [J Biol Chem (1980) 255:9534–9537]. Within 2 h after 1α(OH)D₃ injection in rachitic chicks, there was a significant elevation of 1,25(OH)₂D₃ receptor occupancy in the intestinal mucosa. This observation represents the first direct confirmation that this synthetic analog exerts biological effects through occupancy of 1,25(OH)₂D₃ receptors. Serum 1,25(OH)₂D₃ levels reached a 3-fold higher peak after 1,25(OH)₂D₃ injection (3.25 nmol) than after 1α(OH)D₃ injection (6.5 nmol); further, after 1α(OH)D₃ injection the peak was delayed by 2–4 h. However, serum 1,25(OH)₂D₃ levels remained elevated for only 3–6 h after 1,25(OH)₂D₃, compared to 48 h after 1α(OH)D₃ injection. Occupied 1,25(OH)₂D₃ receptor levels paralleled serum 1,25(OH)₂D₃ levels at all times after administration of either steroid. At 24 h, duodenal vitamin D-dependent calcium binding protein (CaBP) levels were similarly elevated in both treatment groups, but by 48 and 72 h after 1α(OH)D₃ administration CaBP and serum Ca²⁺, respectively, were more significantly elevated. These data confirm that 1α(OH)D₃ induces its major biological effects via intracellular 1,25(OH)₂D₃ receptors and reinforce the concept that 25-hydroxylation is a prerequisite for these effects. These results also suggest that 1α(OH)D₃ may become useful in the therapy for sustained treatment of vitamin D deficiency diseases.     

Induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin D3

Summary 1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin la (IL-1). In order to reveal how 1,25(OH)₂D₃ interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in a-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)₂D₃ enhanced the M-CSF's effect on expression of both antigens, although (1,25(OH)₂D₃) per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)₂D₃ and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)₂D₃ and M-CSF significantly enhanced phagocytic activity and H₂O₂ production, whereas successive treatment with (1,25(OH)₂D₃) and GM-CSF significantly enhanced only phagocytic activity. Enzymehistochemical study demonstrated that cells treated simultaneously or successively with 1,25(OH)₂D₃ and M-CSF were strongly positive for nonspecific esterase (NSE), a macrophage-specific marker, and that simultaneous or successive treatment with 1,25(OH)₂D₃ and GM-CSF yielded cells strongly positive for NSE or for chloroacetate esterase (ChAE), a granulocyte-specific marker, respectively. These findings suggest that 1,25(OH)₂D₃ primes bone marrow progenitor cell populations not only to M-CSF but also to GM-CSF and thereby accelerates the CSFs-dependent differentiation of the cells to the macrophage or granulocyte.     

Effects of increased doses of 1,25 dihydroxyvitamin D3 on matrix and DNA synthesis in condylar cartilage of suckling mice

Summary Thein vivo effects of high doses of 1,25(OH)₂D₃ were studied in condylar cartilage of suckling mice. Seven-day-old animals were treated with 20 ng of the hormone for 7 consecutive days. Biochemical assays on collagen content and synthesis were complemented by structural studies using light and electron microscopy. Indirect immunofluorescent methods were used for the localization of type I and II collagens and for fibronectin. This study revealed that the protein content of the condyle decreased substantially following the administration of the hormone. Protein synthesis increased in hormone-treated animals during the first 4 days but was significantly inhibited theeafter. Collagen synthesis, however, was inhibited instantaneously, followed by a decrease in the percentage of cold hydroxyproline of the total protein. Hormone-treated condyles showed a marked decrease in the distribution of type I collagen, no apparent change in the distribution of type II collagen, but an enhanced reactivity for fibronectin especially around hypertrophic chondrocytes. SDS-gel electrophoresis of collagen chains suggested that the hormone did not induce a significant change in the ratios of type I and II collagen chains, yet additional peaks became evident in 1,25(OH)₂D₃-treated specimens. The decrease in collagen synthesis was accompanied by ultrastructural changes in the appearance of the extracellular collagen bundles. They later appeared as a dense meshwork of collagen fibrils, a feature that was lacking in control tissues. The changes in collagen fibrillogenesis could be explained by ourin vitro studies indicating a marked depression of³⁵S-sulfate incorporation secondary to treatment with 1,25(OH)₂D₃. The hormone was also found to suppress the incorporation of³H-thymidine, hence it may be concluded that 1,25(OH)₂D₃, when used in high concentrations, possesses an inhibitory effect upon both the proliferative activity of the cartilage progenitor cells as well as upon the metabolic activity of the condylar cells as related to collagen and glycosaminoglycans synthesis.     

Effect of vitamin D on growth cartilage cell proliferation in vitro

Disturbed calcification of the growth plate and stunting is a frequent finding in vitamin D-deficiency rickets, vitamin D-dependency rickets and renal osteodystrophy, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of Ic, 25-dihydroxy vitamin D₃ [1,25(OH)₂D₃] receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In tibial growth plates and in primary cultures of tibial growth cartilage of male Sprague-Dawley rats (80 g) specific binding of [³H]-1,25(OH)₂D₃ was noted. Scatchard analysis revealed the presence of a single class of non-interacting binding sites.K _d was 10^–11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA-cellulose affinity chromatography. By immunohistology, growth cartilage cells (rabbit tibia) were shown to express nuclear 1,25(OH)₂D₃ receptors, most prominently in the proliferative and early hypertrophic zone. This corresponds to binding data which showed highest binding of 1,25(OH)₂D₃ in the logarithmic growth phase (12,780 molecules/cell versus 4,538 molecules/cell in confluent cells) in primary cultures of growth plate chondrocytes. In the presence of delipidated fetal calf serum 1,25(OH)₂D₃ had a biphasic effect on cell proliferation and density, i.e. stimulation at 10^–12 M and dose-dependent inhibition at 10^–10 M and below. Inhibition was specific and not seen with 24 (R), 25-dihydroxyvitamin D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and specific effects of 1,25(OH)₂D₃ on chrondrocyte proliferation in vitro point to a role for vitamin D in the homeostasis of growth cartilage of the rat.