Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog

Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.     

The classic receptor for 1alpha,25-dihydroxy vitamin D3 is required for non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in osteosarcoma cells

1alpha,25-dihydroxy vitamin D3 has a major role in the regulation of the bone metabolism as it promotes the expression of key bone-related proteins in osteoblastic cells. In recent years it has become increasingly evident that in addition to its well-established genomic actions, 1alpha,25-dihydroxy vitamin D3 induces non-genomic responses by acting through a specific plasma membrane-associated receptor. Results from several groups suggest that the classical nuclear 1alpha,25-dihydroxy vitamin D3 receptor (VDR) is also responsible for these non-genomic actions of 1alpha,25-dihydroxy vitamin D3. Here, we have used siRNA to suppress the expression of VDR in osteoblastic cells and assessed the role of VDR in the non-genomic response to 1alpha,25-dihydroxy vitamin D3. We report that expression of the classic VDR in osteoblasts is required to generate a rapid 1alpha,25-dihydroxy vitamin D3-mediated increase in the intracellular Ca(2+) concentration, a hallmark of the non-genomic actions of 1alpha,25-dihydroxy vitamin D3 in these cells.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Molecular basis of the selective activity of vitamin D analogues

More than 2,000 synthetic analogues of the biological active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), are presently known. Basically, all of them interfere with the molecular switch of nuclear 1alpha,25(OH)(2)D(3) signaling, which is the complex of the vitamin D receptor (VDR), the retinoid X receptor (RXR), and a 1alpha,25(OH)(2)D(3) response element (VDRE). Central element of this molecular switch is the ligand-binding domain (LBD) of the VDR, which can be stabilized by a 1alpha,25(OH)(2)D(3) analogue either in its agonistic, antagonistic, or non-agonistic conformation. The positioning of helix 12 of the LBD is of most critical importance for these conformations. In each of the three conformations, the VDR performs different protein-protein interactions, which then result in a characteristic functional profile. Most 1alpha,25(OH)(2)D(3) analogues have been identified as agonists, a few are antagonists (e.g., ZK159222 and TEI-9647), and only Gemini and some of its derivatives act under restricted conditions as non-agonists. The functional profile of some 1alpha,25(OH)(2)D(3) analogues, such as EB1089 and Gemini, can be modulated by protein and DNA interaction partners of the VDR. This provides them with some selectivity for DNA-dependent and -independent signaling pathways and VDRE structures.     

The effect of FokI vitamin D receptor polymorphism on bone mineral density in Jordanian perimenopausal women

CONTEXT:

Osteoporosis is a polygenic, multifactorial disease that is characterized by demineralization of bone, and thus presented with decreasing bone mineral mass. Vitamin D receptor (VDR) gene polymorphisms in the 3’-end region (as determined by the enzymes BsmI and ApaI) have been inconsistently associated with bone mineral mass. Another important VDR start codon polymorphism (as determined by the enzyme FokI) has been found to be related to adult bone mineral density (BMD) in pre-and post-menopausal American women.

AIMS:

This study aims to investigate the prevalence of the FokI VDR gene polymorphism in Jordanian perimenopausal women and study its relationship with bone mineral density.

MATERIALS AND METHODS:

DNA was isolated from 90 controls (Mean age = 50.41 ± 1.29 y), and 120 patients with symptomatic vertebral fractures (Mean age = 49.14 ± 3.19 y). Restriction Fragment Length Polymorphism (RFLP) analysis of FokI was performed on DNA samples.

STATISTICAL ANALYSIS:

Data was analyzed using SPSS v19 and Microsoft Excel 2007.

RESULTS:

The results showed that in controls, the FF (−0.70 ± 0.51) genotype is associated with high lumbar spine BMD Z-score as compared to Ff (−1.25 ± 0.26) and ff (−1.66 ± 0.47) genotypes (P = 0.0095). In patients, the ff genotype was associated with lower lumbar spine BMD in T-score (−2.31 ± 0.17) and Z-score (−1.56 ± 0.09) genotypes (P = 0.031). No significant association was seen in the femoral neck BMD.

CONCLUSION:

FokI polymorphism may be associated with low BMD in our studied population; however, further studies including other polymorphisms and large sample number are needed.     

Rationale for active vitamin D and analogs in the treatment of osteoporosis

In 1981, Chugai Pharmaceutical succeeded in marketing alfacalcidol, a prodrug of calcitriol, as a therapeutic agent for renal osteodystrophy. In 1983, Chugai succeeded in extending the application of alfacalcidol to the treatment of osteoporosis as well. Clinicians in Japan have accepted alfacalcidol as a remedy for osteoporosis. However, the use of calcitriol and its analogs for the treatment of osteoporosis is still controversial. Some misunderstandings exist internationally about the efficacy of the active form of vitamin D for the treatment of osteoporosis. It is important to emphasize that patients with osteoporosis have intestinal calcium malabsorption and dysfunction in renal activation of vitamin D. When massive doses of parent vitamin D were administered to OVX rats, bone mass increased, but surprisingly, many porotic area were observed in the cortical bone. On the other hand, administration of alfacalcidol increased physiological bone without porotic observation. It is necessary to give the active form of vitamin D, D-hormone, with an RDA-equivalent supply of calcium. Alfacalcidol forms physiological strong bones that are hardly fractured by regulating calcium and bone metabolism. We proposed a new vitamin D analog, 2beta (3-hydroxypropoxy)calcitriol [ED-71] as a therapeutic drug for osteoporosis, which is more potent than calcitriol. ED-71 is now being investigated in phase 2 clinical studies in Japan. ED-71 will appear as more improved drugs for osteoporosis until 2010.     

The importance of nuclear import in protection of the vitamin D receptor from polyubiquitination and proteasome‐mediated degradation

Others and we previously showed that the vitamin D receptor (VDR) is subject to degradation by the 26S proteasome and that treatment with 1,25‐dihydroxyvitamin D₃ (1,25D₃) inhibited this degradation. In the present study, we found that in osteoblasts, but not in intestinal epithelial cells, the VDR was susceptible to degradation by the 26S proteasome. The subcellular site for degradation of the VDR in osteoblasts is the cytoplasm and the site for ligand‐dependent protection of the VDR from the 26S proteasome is the chromatin. These direct relationships between nuclear localization and protection of the VDR from 26S proteasome degradation led us to hypothesize that the unoccupied cytoplasmic VDR is a substrate for polyubiquitination, which targets VDR for degradation by the 26S proteasome, and that nuclear localization has the ability to protect the VDR from polyubiquitination and degradation. To test these hypotheses, we used Cos‐1 cells transfected with human VDR and histidine‐tagged ubiquitin expression vectors. We found that unoccupied VDR was polyubiquitinated and that 1,25D₃ inhibited this modification. Mutations in the nuclear localization signal of VDR (R49W/R50G and K53Q/R54G/K55E) or in the dimerization interface of VDR with retinoid X receptor (M383G/Q385A) abolished the ability of 1,25D₃ to protect the VDR from polyubiquitination, although these mutations had no effect on the ligand‐binding activity of VDR. Therefore, we concluded that in some cellular environments unoccupied cytoplasmic VDR is susceptible to polyubiquitination and proteasome degradation and that ligand‐dependent heterodimerization and nuclear localization protect the VDR from these modifications. J. Cell. Biochem. 110: 926–934, 2010. © 2010 Wiley‐Liss, Inc.