The effects of 1alpha,24(S)-dihydroxyvitamin D(2) analog on cancer cell proliferation and cytokine expression

It is well established that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D, plays a role in regulating proliferation and differentiation of cells, in addition to its classic function in mineral homeostasis. Recent studies have also provided evidence for the involvement of 1alpha,25(OH)(2)D(3) in regulating the immune system. However, therapeutic application of 1alpha,25(OH)(2)D(3) to hyperproliferative diseases such as cancer, or for immunologic purposes, is thwarted by its hypercalcemic activity. In order to overcome this obstacle, analogs of 1alpha,25(OH)(2)D(3) have been produced that exhibit decreased hypercalcemic activity while retaining the growth and immunologic regulating properties. In the present study, the efficacy of 1alpha,24(S)-dihydroxyvitamin D(2) (1alpha,24(S)(OH)(2)D(2)), a vitamin D(2) analog, in restraining cell proliferation was compared to that of 1alpha,25(OH)(2)D(3). In parallel studies, cancer cell lines were grown in increased concentrations (10(-10)-10(-7) M) of each compound for various incubation periods (1-4 days). Growth was assessed by measuring [(3)H]thymidine incorporation. The results revealed that 1alpha,24(S)(OH)(2)D(2) significantly inhibits proliferation to an extent similar to that observed for 1alpha,25(OH)(2)D(3). Moreover, incubating the human leukemia cell line, HL-60, with 1alpha,24(S)(OH)(2)D(2) resulted in an induction of differentiation of these promyelomonocyte cells into monocyte-macrophage-like cells, in a manner similar to that observed with 1alpha,25(OH)(2)D(3). Using a Western procedure, it was also shown that 1alpha,24(S)(OH)(2)D(2) like 1alpha,25(OH)(2)D(3) enhances the expression of vitamin D receptors (VDR) in the rat osteosarcoma cell line, ROS 17/2.8. The expression of tumor necrosis factor (TNF) alpha (TNF-alpha) in human peritoneal macrophages (HPM) obtained from uremic patients treated with continuous ambulatory peritoneal dialysis (CAPD) was found to be regulated by 1alpha,25(OH)(2)D(3) as well as by 1alpha,24(S)(OH)(2)D(2). Incubations of HPM with 1alpha,25(OH)(2)D(3) or 1alpha,24(S)(OH)(2)D(2), have inhibited the expression of TNF-alpha on both mRNA and protein levels. These results suggest that 1alpha,25(OH)(2)D(3) has a role in controlling the rate of inflammation in the peritoneal cavity of CAPD treated patients. Since 1alpha,24(S)(OH)(2)D(2) does not cause hypercalcemia, the present results encourage the possible use of this vitamin D(2) analog in the treatment of cancer and hyper-inflammatory diseases.     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.     

Metabolism of vitamin D(3) by human CYP27A1

Human vitamin D(3) 25-hydroxylase (CYP27A1) cDNA was expressed in Escherichia coli, and its enzymatic properties were revealed. The reconstituted system containing the membrane fraction prepared from the recombinant E. coli cells was examined for the metabolism of vitamin D(3). Surprisingly, at least eight forms of metabolites including the major product 25(OH)D(3) were observed. HPLC analysis and mass spectrometric analysis suggested that those metabolites were 25(OH)D(3), 26(OH)D(3), 27(OH)D(3), 24R,25(OH)(2)D(3), 1alpha, 25(OH)(2)D(3, )25,26(OH)(2)D(3) (25,27(OH)(2)D(3)), 27-oxo-D(3) and a dehydrogenated form of vitamin D(3). These results suggest that human CYP27A1 catalyzes multiple reactions and multiple-step metabolism toward vitamin D(3). The K(m) and V(max) values for vitamin D(3) 25-hydroxylation and 25(OH)D(3) 1alpha-hydroxylation were estimated to be 3.2 microM and 0.27 (mol/min/mol P450), and 3.5 microM and 0.021 (mol/min/mol P450), respectively. These kinetic studies have made it possible to evaluate a physiological meaning of each reaction catalyzed by CYP27A1.     

Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).     

Potent inhibition of dendritic cell differentiation and maturation by vitamin D analogs

We show that the immunosuppressive effects of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are due, in part, to inhibition of the T cell stimulatory functions of dendritic cells (DCs). Addition of 10(-12) and 10(-8) M 1alpha,25(OH)(2)D(3) to murine DC cultures resulted in a concentration-dependent reduction in levels of class II MHC and the co-stimulatory ligands B7-1, B7-2, and CD40 without affecting the number of DCs generated. Higher concentrations of 1alpha,25(OH)(2)D(3) reduced DC yield. The capacity of DCs to induce proliferation of purified allogeneic T cells was reduced by 1alpha,25(OH)(2)D(3). The vitamin D(3) analog, 1alpha,25(OH)(2)-16-ene-23-yne-26,27-hexafluoro-19-nor -D(3), exerted identical effects at 100-fold lower concentrations. Inhibition of DC maturation and stimulatory function was absent in cultures from mice genetically lacking vitamin D receptors (VDR). Vitamin D analogs effectively reduce DC function via VDR-dependent pathways.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Inhibition by 1 alpha,25-dihydroxyvitamin D3 of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Regulation of erythroid differentiation by vitamin D3 derivatives was examined in Friend erythroleukemia cells. After Friend cells were cultured for 5 days with 1.5% dimethyl sulfoxide (DMSO), as much as 70% of the cells became benzidine-positive and the hemoglobin content increased in parallel with the increase of benzidine-positive cells. The DMSO-induced erythroid differentiation was markedly inhibited by concurrent addition of the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent in inhibiting DMSO-induced erythroid differentiation. 1 alpha,25(OH)2D3 alone was totally ineffective in both cell growth and erythroid differentiation. These results together with our previous reports indicate that 1 alpha,25(OH)2D3 is somehow involved not only in myeloid differentiation, but also in erythroid differentiation.     

Novel vitamin D3 derivatives, 26-homo-delta 22-dehydro-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-dehydro-1 alpha,25(R)-dihydroxyvitamin D3: preferential activity in c-myc mRNA production and in induction of phenotypic differentiation of HL-60 cells

Novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-1 alpha,25(R)-dihydroxyvitamin D3 were compared with the native hormone, 1,25-dihydroxyvitamin D3, and with other vitamin D3 derivatives, in inhibition of cell growth, induction of phenotypic differentiation, and c-myc mRNA reduction of HL-60 cells. The degree of inhibition in cell growth caused by 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 was the greatest followed by 26-homo-delta 22-1 alpha,25(R)-(OH)2D3. The ability to reduce NBT was parallel to that to inhibit cellular proliferation. 26-homo-delta 22-1 alpha,25(S)-(OH)2D3, 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, 24-homo-24-F2-1 alpha,25-(OH)2D3, and 1 alpha,24(R)-(OH)2-26-Cl-D3 were more active than 1 alpha,25-(OH)2D3 in the induction of OK-M1+ and OK-Mo-2+ HL-60 cells. Using two color flow cytometric analysis, the percentages of OK-M5(+)- and OK-DR(+)-HL-60 cells were 33% in the treatment with 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 plus interferon-gamma (IFN-gamma) but 14% in the treatment with 1 alpha,25-(OH)2D3 plus IFN-gamma. 26-Homo-delta 22-1 alpha,25(S)-(OH)2D3 has an inhibitory effect on c-myc reduction in treated HL-60 cells. These results suggest that the novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 and 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, have preferential activity in inducing phenotypic differentiation and in inducing cell proliferation related c-myc mRNA activity.     

Physiological significance of C-28 hydroxylation in the metabolism of 1alpha,25-dihydroxyvitamin D(2).

In our previous study, we indicated for the first time that C-28 hydroxylation plays a significant role in the metabolism of 1alpha, 25-dihydroxyvitamin D(2) [1alpha,25(OH)(2)D(2)] by identifying 1alpha,24(S),25,28-tetrahydroxyvitamin D(2) [1alpha,24(S),25, 28(OH)(4)D(2)] as a major renal metabolite of 1alpha,25(OH)(2)D(2) [G. S. Reddy and K-Y. Tserng Biochemistry 25, 5328-5336, 1986]. The present study was performed to establish the physiological significance of C-28 hydroxylation in the metabolism of 1alpha, 25(OH)(2)D(2). We perfused rat kidneys in vitro with 1alpha, 25(OH)(2)[26,27-(3)H]D(2) (5 x 10(-10)M) and demonstrated that 1alpha,24(R),25-trihydroxyvitamin D(2) [1alpha,24(R),25(OH)(3)D(2)] and 1alpha,24(S),25,28(OH)(4)D(2) are the only two major physiological metabolites of 1alpha,25(OH)(2)D(2). In the same perfusion experiments, we also noted that there is no conversion of 1alpha,25(OH)(2)D(2) into 1alpha,25,28-trihydroxyvitamin D(2 )[1alpha,25,28(OH)(3)D(2)]. Moreover, 1alpha,24(S),25,28(OH)(4)D(2) is not formed in the perfused rat kidney when synthetic 1alpha,25, 28(OH)(3)D(2) is used as the starting substrate. This finding indicates that C-28 hydroxylation of 1alpha,25(OH)(2)D(2) occurs only after 1alpha,25(OH)(2)D(2) is hydroxylated at C-24 position. At present the enzyme responsible for the C-28 hydroxylation of 1alpha, 24(R),25(OH)(3)D(2) in rat kidney is not known. Recently, it was found that 1alpha,25(OH)(2)D(3)-24-hydroxylase (CYP24) can hydroxylate carbons 23, 24, and 26 of various vitamin D(3) compounds. Thus, it may be speculated that CYP24 may also be responsible for the C-28 hydroxylation of 1alpha,24(R),25(OH)(3)D(2) to form 1alpha, 24(S),25,28(OH)(4)D(2). The biological activity of 1alpha,24(S),25, 28(OH)(4)D(2), determined by its ability to induce intestinal calcium transport and bone calcium resorption in the rat, was found to be almost negligible. Also, 1alpha,24(S),25,28(OH)(4)D(2) exhibited very low binding affinity toward bovine thymus vitamin D receptor. These studies firmly establish that C-28 hydroxylation is an important enzymatic reaction involved in the inactivation of 1alpha,25(OH)(2)D(2) in kidney under physiological conditions.     

Evaluation of C-2-substituted 19-nor-1alpha,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1alpha,25(OH)(2)D(3) can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized (3)H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1alpha,25(OH)(2)D(3) analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2alpha- or 2beta-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1alpha,25(OH)(2)D(3). The (3)H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1alpha,25(OH)(2)D(3), 19-nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) or 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) for 7 days. 19-Nor-2alpha-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) and 19-nor-2beta-(3-hydroxypropyl)-1alpha,25(OH)(2)D(3) were also shown to be about 10-fold more active than 1alpha,25(OH)(2)D(3) in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1alpha,25(OH)(2)D(3) molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

Metabolites and analogs of 1alpha,25-dihydroxyvitamin D(3): evaluation of actions in bone

Analogs of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] activate both genomic mechanisms via the nuclear vitamin D(3) receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D(3) receptor (pmVDR). Both of these pathways are normally activated by 1alpha,25(OH)(2)D(3), but as a result of synthesis of numerous analogs of 1alpha,25(OH)(2)D(3) these pathways can be distinguished. We used increasing doses of vitamin D(3) analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D(3) (R) and 1alpha,25(OH)(2)-16,23E-diene-D(3) (A) are 10-fold more potent than 1alpha,25(OH)(2)D(3) for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1alpha-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1alpha,25(OH)(2)D(3) to increase osteocalcin mRNA or protein release. 1alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D(3), (E) is an agent that is 10 fold more potent than 1alpha,25(OH)(2)D(3) at increasing osteocalcin mRNA and protein release, whereas 1alpha,25(OH)(2)-3-epi-D(3) increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1alpha,25(OH)(2)D(3). These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F(6) on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1alpha-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.