Modulation of response to estrogens in cultured human female bone cells by a non-calcemic Vitamin D analog: changes in nuclear and membranal binding

Estradiol17beta (E2) and the phytoestrogens genistein (G), and daidzein (D) increase creatine kinase (CK) specific activity in primary cell cultures of human female to a greater extent in cells from pre-menopausal than post-menopausal women. Pretreatment with the non-calcemic analog of Vitamin D, JK 1624 F2-2 (JKF), upregulated this estrogenic response at all ages. In contrast, biochainin A (BA) and quercertin (Qu) increased CK with no age dependence or modulation by JKF pretreatment. Both ERalpha and ERbeta present in the cells were upregulated by pretreatment with JKF, as measured by Western blot analysis. Real time PCR showed no significant change in ERalpha mRNA but a marked decrease in ERbeta mRNA in both age groups after JKF treatment. Cells from both age groups had surface binding sites for E2, shown by assays using cell impermeable Europium labeled ovalbumin-E2 conjugate (Eu-Ov-E2). Binding of [3H]-E2 to intracellular E2 receptors (ERs) was similar in both age groups with differences in phytoestrogenic competition. JKF pretreatment increased nuclear but decreased membranal binding in both age groups. These results provide evidence for membranal, in addition to nuclear estrogen receptors which are differentially modulated by a Vitamin D analog.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

Culture serum-induced conversion from agonist to antagonist of a Vitamin D analog, TEI-9647

The nuclear receptor for Vitamin D (VDR) mediates many of the effects of Vitamin D in target tissues by regulating gene expression. The transactivation function of ligand-bound VDR in target tissues is thought to depend on the tissue-type and the cellular-environment, but the molecular basis for these differences has not been fully understood. In this study, during characterization of TEI-9647 as a synthetic ligand for the VDR, we found that depletion of serum from the culture medium converted TEI-9647 from an antagonist to an agonist of VDR-mediated transactivation, whereas it retained antagonistic activity in the presence of serum. Consistent with these results, using a mammalian two-hybrid system, we found that TEI-9647 recruited different coactivators to the VDR in the presence and absence of serum. These findings suggest that an unknown serum factor modulates the transactivation function of the VDR.     

Effects of a synthetic analog of polycavernoside A on human neuroblastoma cells.

BACKGROUND: Polycavernoside A is a glycosidic marine toxin first extracted from the red alga Polycavernosa tsudai in 1991 when 3 people died after the ingestion of this food. Polycavernoside A is an interesting molecule because of its complex macrolide structure and strong bioactivity. However, the target site of this toxin has not been characterized. METHODS: We studied the effects of a synthethic analog of polycavernoside A on human neuroblastoma cells by measuring changes in membrane potential with bis-oxonol and variations in intracellular calcium levels with fura-2. Fluorescent phalloidin was utilized for assaying activity on actin cytoskeleton. RESULTS: Data showed that this polycavernoside A analog induced a membrane depolarization and an increase in cytosolic calcium levels. CONCLUSION: These results provide the first insight into the mode of action of polycavernoside A, suggesting that: i) this toxin triggers an initial extracellular calcium entry neither produced across L-type voltage-gated calcium channels nor activation of muscarinic receptors ii) there is a depolarization induced by the toxin and due to the extracellular calcium entry.     

Comparison of the mineralocorticoid activity of 19-oxygenated and 19-nor derivatives of deoxycorticosterone.

19-Nordeoxycorticosterone (19-nor-DOC) is a mineralocorticoid with several unresolved physiologic questions. First, is 19-nor-DOC synthesized in the kidney from a circulating adrenocortical precursor (19-oicdeoxycorticosterone [19-oic-DOC] or 19-oxodeoxycorticosterone [19-oxo-DOC])? Second, does 19-nor-DOC, synthesized in the kidney, have mineralocorticoid activity or is it excreted in the urine without biologic activity? To answer this question, we administered two of the putative 19-nor-DOC precursors (19-oxo-DOC and 19-oic-DOC) to adrenalectomized rats and measured the formation of 19-nor-DOC and bioactivity as the urinary Na+ to K+ ratio. Each of the 10-microgram steroid treatments produced an elevation of urinary-free 19-nor-DOC (0 to 2 hours), whereas at the 1-micrograms dose only 19-oic-DOCA produced an increased UF 19-nor-DOC. None of the treatments led to an increase of conjugated 19-nor-DOC except 10 microgram 19-oic-DOCA. Increased mineralocorticoid activity (decreased urinary Na+ to K+ ratio) was produced by aldosterone, 1 and 10 micrograms 19-nor-DOC, and 10 micrograms 19-oic-DOCA over the same time period. An anti-mineralocorticoid effect (increased urinary Na+ to K+ ratio) was produced by 1 microgram 19-oxo-DOC. Urinary-free 19-nor-DOC, but not conjugated 19-nor-DOC, correlated with the urinary mineralocorticoid effect (decreased Na+ to K+ ratio). These data support the contention that 19-oic-DOC is the circulating 19-nor-DOC precursor and that, at least at the higher dose, it has a mineralocorticoid action on the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of norethynodre, a 19-nor progestin: subcellular localization of enzyme activity

The subcellular distribution of the enzymes involved in the metabolism of norethynodrel (17 alpha-ethynyl-17 beta-hydroxy-estr-5(10)-en-3-one) to the 3alpha and 3beta diols (17 alpha-ethynyl-3alpha (or 3beta-17 beta-dihydroxy-estr-5(10)-ene) and 17 alpha-ethinyl estradiol was studied. The purity of the male rat liver subcellular fractions was evaluated by the use of marker enzymes. Sample sections were viewed by electron microscopy. The data showed that the cytosol fraction contained the highest relative specific activity for the hydroxysteroid dehydrogenases required for the formation of the diols. The cytosol fraction also contained the highest total activity. The enzymes required for the formation of ethinyl estradiol were distributed equally among mitochondrial and microsomal fractions, however, the highest relative specific activity was associated with the heavy microsomal fraction (18,000 g).     

Vitamin D and Its Potential Benefit for the COVID-19 Pandemic

Vitamin D is known not only for its importance for bone health but also for its biologic activities on many other organ systems. This is due to the presence of the vitamin D receptor in various types of cells and tissues, including the skin, skeletal muscle, adipose tissue, endocrine pancreas, immune cells, and blood vessels. Experimental studies have shown that vitamin D exerts several actions that are thought to be protective against coronavirus disease (COVID-19) infectivity and severity. These include the immunomodulatory effects on the innate and adaptive immune systems, the regulatory effects on the renin-angiotensin-aldosterone-system in the kidneys and the lungs, and the protective effects against endothelial dysfunction and thrombosis. Prior to the COVID-19 pandemic, studies have shown that vitamin D supplementation is beneficial in protecting against risk of acquiring acute respiratory viral infection and may improve outcomes in sepsis and critically ill patients. There are a growing number of data connecting COVID-19 infectivity and severity with vitamin D status, suggesting a potential benefit of vitamin D supplementation for primary prevention or as an adjunctive treatment of COVID-19. Although the results from most ongoing randomized clinical trials aiming to prove the benefit of vitamin D supplementation for these purposes are still pending, there is no downside to increasing vitamin D intake and having sensible sunlight exposure to maintain serum 25-hydroxyvitamin D at a level of least 30 ng/mL (75 nmol/L) and preferably 40 to 60 ng/mL (100-150 nmol/L) to minimize the risk of COVID-19 infection and its severity.     

Interactions of various 19-nor steroids with human placental microsomal cytochrome P-450 (P-450hpm).

Each of seven 19-nor steroids exhibited the capacity to facilitate the binding of carbon monoxide (CO) to human placental microsomal cytochrome P-450 although quantitative differences were shown to exist. In every case the facilitation was antagonized by androstenedione. 19-Norandrostenedione produced the most pronounced effect followed by 19-nortestosterone, nandrolone decanoate, norethandrolone, norgestrel, norethynodrel and norethindrone in that order. All steroids investigated produced typical type I binding spectra when added to placental microsomes. Scatched plots also indicated binding of each steroid to two sites--a high-affinity, low-capacity binding site and a low-affinity, high-capacity binding site. Correlations between affinity for either site and capacity to facilitate binding of CO to the cytochrome were not observed nor were there good correlations between maximal absorbance differences (approximately390-420 nm) producible and facilitation capacity. It was therefore concluded that no definitive relationships existed between facilitation capacity and qualitative or quantitative aspects of the steroid-binding spectra. The capacity to facilitate CO binding appeared to reside in the absence of a chemical group substituted at the 10 position on molecules of androgenic steroids since all investigated steroids possessing 10-methyl or other 10-substituted groups either had no effect on the CO-binding spectrum or caused a displacement of CO from ferrous heme. In contrast, all steroids studied that lacked a substitution at C-10 (19-nor steroids) produced a facilitating effect on heme-ligand binding.     

I-labeled 19-iodinated and 6 beta-iodomethyl-19-nor steroids: effect of structural modification on the adrenal accumulation

19-Iodinated and 6 beta-iodomethyl-19-nor derivatives of cholesterol and 17-ketosteroid labeled with 131I were tested in rats to determine the critical structural features required for maximal adrenal uptake. The introduction of the 17-keto group in place of the 17 beta-side chain of cholesterol caused most of the radioactivity to be taken up by the thyroids. Fluorination at the C-3 position had deleterious effects on the adrenal concentration and led to the loss of adrenal specificity. A beta-hydroxy group at the C-3 position is substantially required for adrenal uptake.     

Evaluation of Vitamin D Repletion Regimens to Correct Vitamin D Status in Adults

ObjectiveTo determine the efficacy and safety of commonly prescribed regimens for the treatment of vitamin D insufficiency.MethodsWe performed a retrospective analysis of 306 consecutive patients who were prescribed ergocalciferol (vitamin D₂) for correction of vitamin D insufficiency at the Atlanta Veterans Affairs Medical Center between February 2003 and May 2006. Serum levels of parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and calcium were compared before and after treatment with ergocalciferol. Patients who did not have a 25-OHD determination (n = 41) were excluded from analysis. Vitamin D deficiency, insufficiency, and sufficiency were defined as a serum 25-OHD level of < 20 ng/mL, 21 to 29 ng/mL, and > 30 ng/mL, respectively.ResultsWe identified 36 discrete prescribing regimens. The 3 most common regimens were ergocalciferol 50,000 IU once weekly for 4 weeks followed by 50,000 IU once monthly for 5 months (n = 48); ergocalciferol 50,000 IU once monthly for 6 months (n = 80); and ergocalciferol 50,000 IU 3 times weekly for 6 weeks (n = 27). Each of these 3 treatments significantly increased serum 25-OHD (P < .01), but vitamin D sufficiency was achieved in only 38%, 42%, and 82% of study subjects, respectively. Regimens with > 600,000 IU of ergocalciferol given for a mean of 60 ± 40 days achieved sufficiency in 64% of cases, without vitamin D toxicity.ConclusionIn this study, regimens that contained at least 600,000 IU of ergocalciferol appeared to be the most effective in achieving vitamin D sufficiency. Guidelines for the treatment of vitamin D insufficiency in healthy adults should be developed. (Endocr Pract. 2009;15:95-103)     

Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.     

Anticancer effects of the novel 1alpha, 25-dihydroxyvitamin D3 hybrid analog QW1624F2-2 in human neuroblastoma

Vitamin D3 analogs are potential anti-cancer agents with theoretically wide therapeutic index, but there have been limited studies directed towards human neuroblastoma. The antiproliferative ability of the novel vitamin D3 hybrid analog QW-1624F2-2 (QW, 1-hydroxymethyl-16-ene-24, 24-F2-26, 27-bishomo-25-hydroxyvitamin D3) was examined in two human neuroblastoma-derived cell-lines. Analog QW inhibited cell-cycle progression of IMR5 cells with accumulation in G1 phase. QW induced the differentiation of CHP134 as evidenced by increased neurite length. These effects were accompanied by decreased expression of MYCN in both the cell-lines treated with QW. Furthermore, QW inhibited the migration of CHP134 cells in matrigel invasion assays, indicating its anti-invasive ability. In athymic nude mice, we found that QW was less calcemic than EB1089 (1alpha, 25-dihydroxy-22, 24-diene-24, 26,27-trishomovitamin D3). Systemic administration of QW in a mouse xenotransplantation model revealed that it is more effective than EB1089 in suppressing the growth of CHP134 flank tumors. In summary, the low-calcemic hybrid analog QW showed significant anti-tumor activity in vivo and thus exhibits potential as a novel cancer therapeutic.     

Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death

A chemotherapeutic vitamin D analogue, EB1089, kills tumor cells via a caspase-independent pathway that results in chromatin condensation and DNA fragmentation. Employing transmission- and immunoelectronmicroscopy as well as detection of autophagosome-associated LC3-beta protein in the vacuolar structures, we show here that EB1089 also induces massive autophagy in MCF-7 cells. Interestingly, inhibition of autophagy effectively hindered apoptosis-like nuclear changes and cell death in response to EB1089. Furthermore, restoration of normal levels of beclin 1, an autophagy-inducing tumor suppressor gene that is monoallelically deleted in MCF-7 cells, greatly enhanced the EB1089-induced nuclear changes and cell death. Thus, EB1089 triggers nuclear apoptosis via a pathway involving Beclin 1-dependent autophagy. Surprisingly, tumor cells depleted for Beclin 1 failed to proliferate suggesting that even though the monoallelic depletion of beclin 1 in human cancer cells suppresses EB1089-induced autophagic death, one intact beclin 1 allele is essential for tumor cell proliferation.