1 alpha,25-dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells.

1 alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of the steroid hormone vitamin D, is a potent regulator of macrophage and osteoclast differentiation. The mature osteoclast, unlike the circulating monocyte or the tissue macrophage, expresses high levels of carbonic anhydrase II (CAII). This enzyme generates protons and bicarbonate from water and carbon dioxide and is involved in bone resorption and acid-base regulation. To test whether 1,25(OH)2D3 could induce the differentiation of myelomonocytic precursors toward osteoclasts rather than macrophages, we analyzed its effects on the expression of CAII in bone marrow cultures containing precursors common to both cell types. The expression of CAII was markedly increased by 1,25(OH)2D3 in a dose- and time-dependent manner. In bone marrow, this increase occurred at the mRNA and protein levels and was detectable as early as 24 hr after stimulation. 1,25(OH)2D3 was also found to induce CAII expression in a transformed myelomonocytic avian cell line. These results suggest that 1,25(OH)2D3 regulates the level at which myelomonocytic precursors express CAII, an enzyme that is involved in the function of the mature osteoclast.     

Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts

Osteoblast-osteoclast coordination is critical in the maintenance of skeletal integrity. The modulation of osteoclastogenesis by immature cells of the osteoblastic lineage is mediated through receptor activator of NF kappa B (RANK), its ligand RANKL, and osteoprotegerin (OPG), a natural decoy receptor for RANKL. Here, the expression of OPG and RANKL in primary mouse osteoblastic cultures was investigated to determine whether the osteoclastogenic stimulus depended on the stage of osteoblastic differentiation and the presence of the calciotrophic hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). OPG mRNA expression was increased in osteoblastic cultures after the onset of mineralisation relative to less mature cultures, but did not alter in response to 1,25-(OH)(2)D(3) treatment. In contrast, basal RANK L mRNA expression did not change during differentiation but was significantly enhanced by 1,25-(OH)(2)D(3) treatment at all times. The stimulatory effects of 1,25-(OH)(2)D(3) on RANKL were lessened in more mature cultures, however. The RANKL/OPG ratio, an index of osteoclastogenic stimulus, was therefore increased by 1,25-(OH)(2)D(3) treatment at all stages of osteoblastic differentiation, but to a lesser degree in cultures after the onset of mineralisation. Thus the 1,25-(OH)(2)D(3)-driven increase in osteoclastogenic potential of immature osteoblasts appears to be mediated by increased RANKL mRNA expression, with mature osteoblasts having relatively decreased osteoclastogenic activity due to increased OPG mRNA expression. These findings suggest a possible mechanism for the recently proposed negative regulatory role of mature osteoblasts on osteoclastogenesis and indicate that the relative proportions of immature and mature osteoblasts in the local microenvironment may control the degree of resorption at each specific bone site.     

Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.     

Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10(-10) to 10(-7) M, induces the differentiation of HL60 into mono- and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4- to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of alpha-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.     

Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism.

We describe several novel analogs of the seco-steroid 1,25(OH)2-vitamin D3[1,25(OH)2D3] and their effects on differentiation and proliferation of HL-60 human myeloid leukemic cells in vitro as well as their effects on calcium metabolism in vivo. The 1 alpha-25(OH)2-16ene-23yne-26,27F6-vitamin D3 is the most potent analog reported to date, having about 80-fold more activity than the reference 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of HL-60 cells. Also, this analog decreased RNA expression of MYC oncogene in HL-60 by 90% at 5 x 10(-10) mol/L. Intriguingly, intestinal calcium absorption and bone calcium mobilization mediated in vivo by 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3 was found to be markedly (15-fold) less than that of 1,25(OH)2D3. In addition, 1 alpha-25(OH)2D3 bound to 1,25(OH)2D3 receptors of both HL-60 and intestine more avidly than did 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3. This novel analog may open up new therapeutic strategies for several hematopoietic, skin, and bone abnormalities and may provide a new tool to understand how vitamin D3 seco-steroids induce cellular differentiation.     

Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation.

In vitro translation of cellular poly(A)+ RNA coupled with immunoprecipitation was developed as a technique for characterizing 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and assessing receptor mRNA activity. Cell-free translation of poly(A)+ RNA isolated from chicken intestine revealed two immunoprecipitable forms of avian receptor at 60 kDa and 58 kDa. These two species were identical in electrophoretic mobility to those detected directly in intestinal cytosol by immunoblot analysis. Liver, a tissue devoid of 1,25-(OH)2D3 binding activity, contained no apparent translatable receptor mRNA. 1,25-(OH)2D3 receptors were also synthesized in vitro employing poly(A)+ RNA obtained from several cultured mammalian cell lines. Selective immunoprecipitation revealed a single form of receptor at 54 kDa in mouse fibroblasts (3T6) and pig kidney cells (LLC-PK1) and a 52-kDa species in human breast carcinoma (T47D). Each of these in vitro translated mammalian 1,25-(OH)2D3 receptors migrated identically with its cellular counterpart that was synthesized in vivo employing metabolic labeling of cell protein with [35S]methionine. In vitro translation of poly(A)+ RNA derived from mouse 3T6 cells treated with 1,25-(OH)2D3 for 24-48 hr disclosed a 5-fold increase in receptor mRNA activity over untreated control cells. These results are consistent with the conclusions that 1,25-(OH)2D3 receptors are protein species ranging from 52 to 60 kDa and that, though their functional and immunological domains have been evolutionarily conserved, an inverse relationship apparently exists between phylogenetic status and receptor mass. The data also support the hypothesis that the presence of 1,25-(OH)2D3 leads to a significant increase in receptor mRNA activity in 3T6 cells, indicative of receptor autoregulation.     

Effects of 1,25-dihydroxycholecalciferol and calcium on calcitonin mRNA levels in suckling rats

The chronic administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) to 9-day-old suckling rats induced no change on day 13 in the calcitonin (CT) mRNA steady-state level of thyroid glands measured by Northern hybridization. Thyroidal CT contents were decreased in relation to increased plasma calcium levels in animals treated with 0.1 or 1 microgram 1,25-(OH)2D3/kg. Using a lower dose (0.01 microgram/kg), neither plasma calcium, nor thyroidal CT contents were changed. No correlation was found between CT mRNA levels and thyroidal CT contents as well as for plasma CT levels and thyroidal CT contents since hormone in blood remained unchanged after treatment by the active vitamin D3 metabolite. Intraperitoneal calcium administration in fasted 13-day-old rats was associated with a 5-fold increase in plasma CT 30 min after injection, but CT mRNA levels were unchanged within 240 min. By contrast, stomach gavage with calcium in fasted 13-day-old rats induced a sustained increase in plasma CT (X2), and a 4-fold increase in the steady-state level of CT mRNA. Calcium per se is a potent stimulator of CT release in suckling rats, but did not change the amount of CT mRNA. However, gastrointestinal factors may be implied directly or indirectly in the increased CT mRNA level after calcium gavage. In conclusion, 1,25-(OH)2D3 which is known to affect CT gene expression in adult rats is ineffective in 13-day-old suckling rats. This observation may be related to developmental changes in the amount of 1,25-(OH)2D3 receptors of C cells.     

17β-雌二醇和1,25-二羟维生素D3对成骨细胞MC3T3-E1增殖和分化的影响

目的 探讨雌激素和维生素D对成骨细胞MC3T3-E1增殖和分化的协同调节作用及其机制.方法 小鼠成骨细胞系MC3T3-E1细胞用无酚红α-MEM完全培养基培养;用MTT法检测细胞增殖率;用实时荧光定量PCR法检测干预前后MC3T3-E1细胞中细胞周期相关基因[细胞周期素E( cyclin E)、增殖细胞核抗原(PCNA)和细胞周期素依赖性激酶抑制物(Cdkn2b)]和成骨细胞分化标志物[Ⅰ型胶原( COL Ⅰ)、碱性磷酸酶(ALP)和骨桥蛋白(OPN)]基因的表达.ALP活性染色用BCIP/NBT显色法.结果 单用雌激素17β-雌二醇(E2)可促进MC3T3-E1细胞的增殖,尤其在生理浓度时作用最强,单用维生素D活性产物1,25-二羟维生素D3[1,25-(OH) 2D3]对MC3T3-E1细胞的增殖无影响,1,25 - (OH)2 D3不影响E2对MC3T3-E1细胞的促增殖作用.E2上调MC3T3-E1细胞中cyclin E和PCNA的表达,同时下调Cdkn2b基因的表达,1,25-(OH)2D3单独应用不能影响上述基因表达的变化,也不影响E2对上述基因的调节作用.E2可促进MC3T3-E1细胞中分化标志物(COLⅠ、ALP和OPN)基因的表达,加用1,25-(OH)2D3后可增强此作用.结论 雌激素和维生素D作为两种重要的调节骨代谢的激素,在促进成骨细胞增殖方面可能无协同作用,而在促进其分化方面可能有协同作用.     

Cooperative effects of gamma interferon and 1-alpha,25-dihydroxyvitamin D3 in inducing differentiation of human promyelocytic leukemia (HL-60) cells

Various agents induce differentiation of human leukemia cells in vitro. Most of these agents cause myeloid differentiation, but phorbol diesters, 1-alpha,25-dihydroxyvitamin D3 (1,25[OH2]D3), and certain lymphokines cause differentiation to monocyte-like cells. The purpose of this study was to determine the cooperative effects of 1,25(OH2)D3 and the lymphokine gamma interferon (IFN-gamma) on HL-60 cell differentiation. The recombinant human IFN-gamma or 1,25(OH2)D3 caused a slight reduction in the proliferation of the HL-60 cells (30%-40% reduction at doses of 100-200 U/ml [0.25-0.50 nM] IFN-gamma, or 5-25 nM 1,25[OH2]D3). HL-60 cells treated with 100 U/ml IFN-G had an eightfold increase in expression of nonspecific esterase (NSE) and a twofold increase in H2O2 production in response to phorbol myristate acetate (PMA). 1,25(OH2)D3 enhanced NSE expression eight- to 30-fold and H2O2 secretion twofold in response to PMA. There was also enhanced expression of HLA-DR and the receptor for C3bi. The 1,25(OH2)D3- and IFN-gamma-differentiating effects appeared to be additive or synergistic. Populations of IFN-gamma-treated HL-60 cells (but not the 1,25[OH2]D3-treated cells) had multinucleated giant cells. The polykaryons had NSE activity and had some properties of macrophage polykaryons or osteoclasts. 1,25(OH2)D3 did not augment the IFN-gamma-induced polykaryon formation.     

Differentiating agents facilitate infection of myeloid leukemia cell lines by monocytotropic HIV-1 strains [published erratum appears in Blood 1991 Apr 1;77(7):1624]

Monocytotropic human immunodeficiency virus type 1 (HIV-1) isolates from patients with acquired immunodeficiency syndrome (AIDS) infect mononuclear phagocytes as well as activated T cells, but do not usually infect immature human myeloid cell lines in vitro. The HL-60 promyelocytic/myeloblastic cell line and the promonocytic line, U937, were susceptible to productive infection by monocytotropic HIV-1 isolates (HIV-1JR-FL and HTLV-IIIBa-L) after treatment with retinoic acid, dimethyl sulfoxide, dibutyryl cAMP, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Virus production was only detected when these compounds were added before virus infection. Virus replication did not correlate with CD4 receptor expression because undifferentiated HL-60 cells express CD4 and the level of CD4 expression did not increase after differentiation in the presence of retinoic acid, 1,25(OH)2D3, or TPA. A mature monocytic cell line (THP-1) was capable of infection without pretreatment, and treatment with differentiating agents enhanced virus production. A chronically infected cell line (J-HL-60) was isolated after HIV-1JR-FL infection of HL-60 cells treated with retinoic acid. Virus production in this cell line was enhanced more than 10-fold after differentiation in the presence of 1,25(OH)2D3 or TPA. The majority of virus production by 1,25(OH)2D3-treated J-HL-60 cells was associated with the mature, adherent population. Molecular analysis of a cloned line of J-HL-60 showed integration of a single DNA provirus. These results suggest that cellular factors associated with precursor cell differentiation along the myelomonocytic pathway are required for optimal replication of monocytotropic HIV-1 strains in vitro.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Cellular and molecular events associated with the bone-protecting activity of the noncalcemic vitamin D analog Ro-26-9228 in osteopenic rats

We have examined several analogs of 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in an animal model of osteoporosis (ovariectomized rats) to identify a compound with a greater therapeutic range than 1,25-(OH)(2)D(3) for treatment of this bone disease. Here, we report that one analog, Ro-26-9228, had a bone-protecting effect but did not induce hypercalcemia at a wide concentration range. Analysis of biochemical markers and the bone histomorphometry of analog-treated rats suggested that Ro-26-9228 acted by inhibiting bone resorption and increasing the number of differentiated osteoblasts. To determine the basis for the segregation between hypercalcemia and bone-protecting action, we examined gene expression in tissues that regulate calcium homeostasis. We found that 1,25-(OH)(2)D(3) induced 24-hydroxylase mRNA expression in the duodena of ovariectomized rats, but Ro-26-9228 did not. Furthermore, in the duodena of intact animals, 1,25-(OH)(2)D(3) induced a significant increase in calbindin D 9K and plasma membrane calcium pump 1 mRNAs, but Ro-26-9228 had no effect on these mRNAs. On the other hand, the osteoblast-specific gene products osteocalcin and osteopontin were significantly up-regulated in trabecular bone by both the natural hormone and Ro-26-9228. Further investigation of gene-regulatory events in trabecular bone revealed that both 1,25-(OH)(2)D(3) and Ro-26-9228 up-regulated TGF beta1 and beta2 mRNAs. We concluded that the unique properties of Ro-26-9228 include preferential gene regulation in osteoblasts over duodenum and effective induction of growth factors in bone.     

Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone mobilization

Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2-16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.