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Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease have been linked to vitamin D-deficiency. Using a dextran sodium sulphate (DSS)-induced model of IBD we have shown previously that mice raised on vitamin D-deficient diets from weaning have lower serum 25-hydroxyvitamin D (25OHD) levels and develop more severe colitis compared to vitamin D-sufficient counterparts. We have also shown in vitro that immune responses to 25OHD may depend on 'free' rather than total serum concentrations of 25OHD. To investigate the possible effects of free versus total 25OHD on anti-inflammatory immune responses in vivo we have studied DSS-induced colitis in wild type C57BL/6 mice raised from weaning on diets containing vitamin D2 (D2) or vitamin D3 (D3) only (both 1000 IU/kg feed). 25OHD2 has lower binding affinity for the vitamin D binding protein than 25OHD3 which results in higher levels of free 25OHD2 relative to free 25OHD3 in mice raised on a D2-only diet. Total serum 25OHD concentrations, measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showed that D2 mice had significantly lower levels of 25OHD than D3 mice (6.85 ± 2.61 nmol/L vs. 49.16 ± 13.8 nmol/L for D2 and D3 respectively). Despite this, direct ELISA measurement showed no difference in free serum 25OHD levels between D2 and D3 mice (13.62 ± 2.26 pmol/L vs. 14.11 ± 2.24 pmol/L for D2 and D3 respectively). Analysis of DSS-induced colitis also showed no difference in weight loss or disease progression between D2 and D3 mice. These data indicate that despite D2-fed mice being vitamin D-deficient based on serum total 25OHD concentrations, these mice showed no evidence of increased inflammatory colitis disease relative to vitamin D-sufficient D3 mice. We therefore propose that free, rather than total serum 25OHD, may be a better marker of immune responses to vitamin D in vivo.

UnlabelledVitamin D is hypothesized to suppress inflammation. We tested total and free vitamin D metabolites and their association with inflammatory markers. Interleukin-6 levels were lower with higher 25-hydroxyvitamin D. 1,25-dihydroxyvitamin D and free 25OHD associations mirrored those of 25OHD. However, associations for the two metabolites diverged for tumor necrosis factor alpha (TNF-α) soluble receptors.IntroductionVitamin D is hypothesized to suppress inflammation, and circulating 25-hydroxyvitamin D (25OHD) and inflammatory markers are inversely correlated. However, total serum 25OHD may not be the best indicator of biologically active vitamin D.MethodsWe tested serum total 25OHD, total 1,25(OH)2D, vitamin D binding protein (DBP), and estimated free 25OHD and free 1,25(OH)2D associations with inflammatory markers serum interleukin-6 (IL-6), TNF-α and their soluble receptors, interleukin-10 (IL-10), and C-reactive protein (CRP) as continuous outcomes and the presence of ≥2 inflammatory markers in the highest quartile as a dichotomous outcome, in a random subcohort of 679 men in the Osteoporotic Fractures in Men (MrOS) study.ResultsIL-6 was lower in men with higher 25OHD (-0.23 μg/mL per standard deviation (SD) increase in 25OHD, 95 % confidence intervals (CI) -0.07 to -0.38 μg/mL) and with higher 1,25(OH)2D (-0.20 μg/mL, 95 % CI -0.0004 to -0.39 μg/mL); free D associations were slightly stronger. 25OHD and DBP, but not 1,25(OH)2D, were independently associated with IL-6. TNF-α soluble receptors were inversely associated with 1,25(OH)2D but positively associated with 25OHD, and each had independent effects. The strongest association with ≥2 inflammatory markers in the highest quartile was for free 1,25(OH)2D (odds ratios (OR) 0.70, 95 % CI 0.54 to 0.89 per SD increase in free 1,25(OH)2D).ConclusionsAssociations of 1,25(OH)2D and free 25OHD with IL-6 mirrored those of 25OHD, suggesting that 1,25(OH)2D and free D do not improve upon 25OHD in population-based IL-6 studies. However, associations for the two metabolites diverged for TNF-α soluble receptor, warranting examination of both metabolites in studies of TNF-α and its antagonists.

Background: Apart from the well-established skeletal effects, v itamin D has been explored as a secretagogue influencing various adipokines, including adiponectin and irisin. Recent evidence suggests that specific forms of 25-Hydroxyvitamin D (25(OHD), such as free and bioavailable 25(OH)D, may provide more accurate measurements of vitamin D status. The relationship between vitamin D status and serum irisin and adiponectin concentrations remains largely unexplored, particularly during pregnancy., Methods: We analyzed data from 67 healthy maternal-neonatal pairs from Northern Greece at birth. Biochemical and hormonal tests were conducted on each maternal-neonatal pair. The vitamin D forms were estimated using validated mathematical models. Subsequently, regression analyses were conducted to determine the association between the vitamin D forms and adipokine levels., Results: Bioavailable maternal 25(OH)D was inversely associated with neonatal irisin concentrations [β=-73.46 (-140.573 to -6.341), p=0.034]. No other associations were observed between maternal vitamin D status and neonatal adipokine concentrations., Conclusion: In conclusion, maternal bioavailable vitamin D concentrations are inversely associated with neonatal serum irisin concentrations, warranting further studies to evaluate the underlying mechanisms for this finding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Arabi, Fakhoury, Tamim, Chun, Hewison, AlAnouti, Pilz, Annweiler, Tzimagiorgis, Haitoglou and Karras.)

Sufficient vitamin D status is crucial for successful pregnancy and fetal development. The assessment of 25-hydroxyvitamin D (25(OH)D) concentrations is commonly used to evaluate vitamin D status. Our objective was to examine the interrelated biodynamics of maternal and neonatal total, free and bioavailable 25(OH)D in maternal-neonatal dyads at birth and their associations with homeostasis and neonatal birth anthropometry. We analysed a cohort of seventy full-term mother-child pairs. We found positive associations between all neonatal measures of vitamin D status. Maternal forms exhibited a similar pattern of association, except for the bioavailable maternal form. In multivariate analysis, both total and free maternal 25(OH)D concentrations were correlated with all neonatal forms (neonatal total 25(OH)D: 1·29 (95 % CI, 1·12, 1·46) for maternal total 25(OH)D, 10·89 (8·16, 13·63) for maternal free 25(OH)D), (neonatal free 25(OH)D: 0·15 for maternal total 25(OH)D, 1·28 (95 % CI, 0·89, 1·68) for maternal free 25(OH)D) and (0·13 (95 % CI, 0·10, 0·16), 1·06 (95 % CI, 0·68, 1·43) for maternal free 25(OH)D), respectively, with the exclusion of the bioavailable maternal form. We observed no significant interactions within or between groups regarding maternal and neonatal vitamin D parameters and maternal calcium and parathyroid hormone concentrations, and neonatal birth anthropometry. Our study indicates that bioavailable maternal and neonatal 25(OH)D have no significant effects on vitamin D equilibrium, Ca homeostasis and neonatal anthropometry at birth. However, we observed an interaction between maternal and neonatal total and free 25(OH)D concentrations at the maternal-neonatal interface, with no associations observed with other calciotropic or anthropometric outcomes.

Background: Vitamin D deficiency has been associated with worse coronavirus disease 2019 (COVID-19) outcomes, but circulating 25-hydroxyvitamin D [25(OH)D] is largely bound to vitamin D-binding protein (DBP) or albumin, both of which tend to fall in illness, making the 25(OH)D status hard to interpret. Because of this, measurements of unbound ("free") and albumin-bound ("bioavailable") 25(OH)D have been proposed., Objectives: We aimed to examine the relationship between vitamin D status and mortality from COVID-19., Methods: In this observational study conducted in Liverpool, UK, hospitalized COVID-19 patients with surplus sera available for 25(OH)D analysis were studied. Clinical data, including age, ethnicity, and comorbidities, were extracted from case notes. Serum 25(OH)D, DBP, and albumin concentrations were measured. Free and bioavailable 25(OH)D were calculated. Relationships between total, free, and bioavailable 25(OH)D and 28-day mortality were analyzed by logistic regression., Results: There were 472 patients with COVID-19 included, of whom 112 (23.7%) died within 28 days. Nonsurvivors were older (mean age, 73 years; range, 34-98 years) than survivors (mean age, 65 years; range, 19-95 years; P = 0.003) and were more likely to be male (67%; P = 0.02). The frequency of vitamin D deficiency [25(OH)D < 50 nmol/L] was similar between nonsurvivors (71/112; 63.4%) and survivors (204/360; 56.7%; P = 0.15) but, after adjustments for age, sex, and comorbidities, increased odds for mortality were present in those with severe deficiency [25(OH)D < 25 nmol/L: OR, 2.37; 95% CI, 1.17-4.78] or a high 25(OH)D (≥100 nmol/L; OR, 4.65; 95% CI, 1.51-14.34) compared with a 25(OH)D value of 50-74 nmol/L (reference). Serum DBP levels were not associated with mortality after adjustments for 25(OH)D, age, sex, and comorbidities. Neither free nor bioavailable 25(OH)D values were associated with mortality., Conclusions: Vitamin D deficiency, as commonly defined by serum 25(OH)D levels (<50 nmol/L), is not associated with increased mortality from COVID-19, but extremely low (<25 nmol/L) and high (>100 nmol/L) levels may be associated with mortality risks. Neither free nor bioavailable 25(OH)D values are associated with mortality risk. The study protocol was approved by the London-Surrey Research Ethics Committee (20/HRA/2282)., (© Crown copyright 2022.)

Purpose: Recent evidence suggests that improving the transitional care process may reduce 30-day readmissions and hospital length of stay (LOS). The objective of this study was to evaluate the impact of a pharmacist-led transitions-of-care (TOC) program on 30- and 90-day all-cause readmissions and LOS for patients discharged from the hospital acute care setting., Methods: A retrospective cohort study was conducted using a difference-in-difference (DID) approach. Patients who were at least 18 years old with any of the following primary diagnoses were included: acute myocardial infarction, chronic obstructive pulmonary disease, congestive heart failure (CHF), and pneumonia. Outcome measures were all-cause 30- and 90-day readmission and LOS for the index admission., Results: From October 2013 through September 2017, 1,776 patients were discharged from the intervention site, and 2,969 patients were discharged from 3 control sites. Only 33.3% of eligible patients at the intervention site actually received the intervention. The DID analysis showed that the odds ratio (OR) for 30-day readmission was 0.65 [P = 0.035] at the intervention site following TOC program initiation. The OR for 90-day readmission was 0.75 [P = 0.070]. Among all diagnosis groups, the CHF subgroup had the highest proportion of patients who actually received the TOC intervention (57.2%). Within that CHF subgroup, the ORs for 30- and 90-day readmissions were 0.52 [P = 0.056] and 0.47 [P = 0.005], respectively. The mean LOS did not change significantly in either analysis., Conclusion: This pharmacist-led transitional care intervention was associated with significantly decreased inpatient readmissions. The analysis indicates that pharmacist interventions can significantly reduce 30-day readmissions for high-risk populations and 90-day readmissions in patients with CHF., (© American Society of Health-System Pharmacists 2020.)

Vitamin D has a long-established role in bone health. In the last two decades, there has been a dramatic resurgence in research interest in vitamin D due to studies that have shown its possible benefits for non-skeletal health. Underpinning the renewed interest in vitamin D was the identification of the vital role of intracrine or localized, tissue-specific, conversion of inactive pro-hormone 25-hydroxyvitamin D [25(OH)D] to active 1,25-dihydroxyvitamin D [1,25(OH) 2 D]. This intracrine mechanism is the likely driving force behind vitamin D action resulting in positive effects on human health. To fully capture the effect of this localized, tissue-specific conversion to 1,25(OH) 2 D, adequate 25(OH)D would be required. As such, low serum concentrations of 25(OH)D would compromise intracrine generation of 1,25(OH) 2 D within target tissues. Consistent with this is the observation that all adverse human health consequences of vitamin D deficiency are associated with a low serum 25(OH)D level and not with low 1,25(OH) 2 D concentrations. Thus, clinical investigators have sought to define what concentration of serum 25(OH)D constitutes adequate vitamin D status. However, since 25(OH)D is transported in serum bound primarily to vitamin D binding protein (DBP) and secondarily to albumin, is the total 25(OH)D (bound plus free) or the unbound free 25(OH)D the crucial determinant of the non-classical actions of vitamin D? While DBP-bound-25(OH)D is important for renal handling of 25(OH)D and endocrine synthesis of 1,25(OH) 2 D, how does DBP impact extra-renal synthesis of 1,25(OH) 2 D and subsequent 1,25(OH) 2 D actions? Are their pathophysiological contexts where total 25(OH)D and free 25(OH)D would diverge in value as a marker of vitamin D status? This review aims to introduce and discuss the concept of free 25(OH)D, the molecular biology and biochemistry of vitamin D and DBP that provides the context for free 25(OH)D, and surveys in vitro , animal, and human studies taking free 25(OH)D into consideration., (Copyright © 2019 Chun, Shieh, Gottlieb, Yacoubian, Wang, Hewison and Adams.)

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn's disease have been linked to vitamin D-deficiency. Using a dextran sodium sulphate (DSS)-induced model of IBD we have shown previously that mice raised on vitamin D-deficient diets from weaning have lower serum 25-hydroxyvitamin D (25OHD) levels and develop more severe colitis compared to vitamin D-sufficient counterparts. We have also shown in vitro that immune responses to 25OHD may depend on 'free' rather than total serum concentrations of 25OHD. To investigate the possible effects of free versus total 25OHD on anti-inflammatory immune responses in vivo we have studied DSS-induced colitis in wild type C57BL/6 mice raised from weaning on diets containing vitamin D2 (D2) or vitamin D3 (D3) only (both 1000 IU/kg feed). 25OHD2 has lower binding affinity for the vitamin D binding protein than 25OHD3 which results in higher levels of free 25OHD2 relative to free 25OHD3 in mice raised on a D2-only diet. Total serum 25OHD concentrations, measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), showed that D2 mice had significantly lower levels of 25OHD than D3 mice (6.85 ± 2.61 nmol/L vs. 49.16 ± 13.8 nmol/L for D2 and D3 respectively). Despite this, direct ELISA measurement showed no difference in free serum 25OHD levels between D2 and D3 mice (13.62 ± 2.26 pmol/L vs. 14.11 ± 2.24 pmol/L for D2 and D3 respectively). Analysis of DSS-induced colitis also showed no difference in weight loss or disease progression between D2 and D3 mice. These data indicate that despite D2-fed mice being vitamin D-deficient based on serum total 25OHD concentrations, these mice showed no evidence of increased inflammatory colitis disease relative to vitamin D-sufficient D3 mice. We therefore propose that free, rather than total serum 25OHD, may be a better marker of immune responses to vitamin D in vivo., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Vitamin D-deficiency has been linked to inflammatory diseases including rheumatoid arthritis (RA). Studies to date have focused on the impact of serum 25-hydroxyvitamin D3 (25(OH)D3), an inactive form of vitamin D, on RA disease activity and progression. However, anti-inflammatory actions of vitamin D are likely to be mediated at sites of RA disease, namely the inflamed joint, and may involve other vitamin D metabolites notably the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D3). In the current study serum and synovial fluid samples from n = 20 patients with persistent RA and n = 7 patients with reactive arthritis (ReA) were analysed for multiple vitamin D metabolites. Serum data for RA and ReA patients were compared to healthy controls (HC). There was no significant difference between RA or ReA patients relative to HC for 25(OH)D3, 24,25(OH) 2 D3, 1,25(OH) 2 D3 or 25(OH)D2. However, 3-epi-25(OH)D3 was significantly lower in RA and ReA patients compared to HC (p < 0.05). All vitamin D metabolites, apart from 25(OH)D2, were lower in SF compared to serum, and SF 1,25(OH) 2 D3 was unquantifiable in 13/20 RA and 4/7 ReA samples. SF 25(OH)D3, 3-epi-25(OH)D3 and DBP correlated inversely with swollen joint score, and serum 25(OH)D2 and SF DBP correlated directly with C-reactive protein levels. These data indicate that serum 25(OH)D3 provides only limited insight into the role of vitamin D in RA. Alternative serum metabolites such as 3-epi-25(OH) 2 D3, and SF metabolites, notably lack of SF 1,25(OH) 2 D3, may be more closely linked to RA disease severity and progress., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Context: The physiologic role of free 25-hydroxyvitamin D [25(OH)D] in humans is unclear., Objective: To assess whether rise in total vs free 25(OH)D is associated with change in downstream biomarkers of 25(OH)D entry into target cells in kidney and parathyroid: 24,25-dihyroxyvitamin D [24,25(OH)2D] and PTH, respectively., Design: 16-week randomized controlled trial., Intervention: 60 μg (2400 IU)/d of D3 or 20 μg/d of 25(OH)D3., Setting: Academic medical center., Participants: 35 adults age ≥18 years with 25(OH)D levels < 20 ng/mL., Main Outcome Measures: 24,25(OH)2D, 1,25-dihyroxyvitamin D [1,25(OH)2D] and PTH., Results: At baseline, participants [D3 and 25(OH)D3 groups combined] were 35.1 ± 10.6 years. Mean total 25(OH)D, free 25(OH)D, 24,25(OH)2D, and PTH were 16.6 ng/mL, 4.6 pg/mL, 1.3 ng/mL, and 37.2 pg/mL, respectively. From 0 to 4 weeks, rise in only free 25(OH)D was associated with a concurrent 24,25(OH)2D increase [P = 0.03, adjusted for change in 1,25(OH)2D and supplementation regimen] and PTH decrease (P = 0.01, adjusted for change in calcium and supplementation regimen). Between 4 and 8 weeks, and again from 8 to 16 weeks, rises in free and total 25(OH)D were associated with 24,25(OH)2D increase; in contrast, rise in neither total nor free 25(OH)D was associated with PTH decrease during these time periods., Conclusions: Early rise in free 25(OH)D during treatment of vitamin D deficiency was more strongly associated with changes in biomarkers of 25(OH)D entry into target kidney and parathyroid cells, suggesting a physiologic role of free 25(OH)D in humans.

To investigate an immunomodulatory role for vitamin D in pregnancy we used mice raised on vitamin D-sufficient (SUFF), or -deficient (DEF) diets. At embryonic day 14, pregnant mice received intraperitoneal injection of lipopolysaccharide (LPS) or vehicle for 24h, with age-matched non-pregnant mice as controls. In non-pregnant mice, 6 serum analytes (IL-1β, IL-18, MDC/CCL22, MIP-1α/CCL3, EGF, IgA) were lower in DEF mice. In pregnant DEF mice only GH was higher. In non-pregnant mice LPS induced 28 analytes, with 5 (IL-18, IP-10/CXCL10, MCP-1/CCL2, MIP-1β/CCL4, MIP-3β/CCL19) being highest in DEF mice. In pregnant SUFF mice 16 serum analytes increased with LPS, and 6 of these (IP-10/CXCL10, MCP-1/CCL2, SAP, TIMP-1, VCAM-1, vWF) were higher and 1 (GCP-2/CXCL6) lower in DEF mice. Parallel analysis of placental mRNAs showed elevated mRNA for Il-6, Ccl2 and Cxcl10 in placentae from male and female fetuses in LPS-DEF mice. However, LPS-induced expression of Ifnγ, Tnfα, and Cxcl6 was only observed in female placentae from DEF mice. LPS-DEF mice also showed smaller litter sizes relative to control SUFF mice. Numbers of female fetuses per dam were significantly lower for DEF mice with or without LPS challenge. LPS had no effect on numbers of male fetuses from DEF mothers, but significantly decreased male fetuses from SUFF mothers. These data indicate that vitamin D is an important component of anti-inflammatory immune responses during pregnancy, with the placenta and fetal sex playing pivotal roles in this process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Despite recent advances, infection remains the most common etiology of arthroplasty failure. Recent work suggests that 25-hydroxyvitamin D (25D) deficiency correlates with the frequency of periprosthetic joint infection (PJI). We endeavored to examine whether 25D3 deficiency leads to increased bacterial burden in vivo in an established mouse model of PJI and, if so, whether this effect can be reversed by preoperative 25D3 supplementation., Methods: Mice (lys-EGFP) possessing fluorescent neutrophils were fed a vitamin D3-sufficient (n = 20) or deficient (n = 40) diet for 6 weeks. A group of 25D3-deficient mice (n = 20) were "rescued" with 1 intraperitoneal dose of 25D3 at 3 days before surgery. A stainless steel implant was inserted into the knee joint and the joint space was inoculated with bioluminescent Staphylococcus aureus (1 × 10 colony forming units [CFUs]). In vivo imaging was used to monitor bacterial burden and neutrophil infiltration. Blood was drawn to confirm 25D3 levels 3 days before surgery and on postoperative days (PODs) 0 and 14. Mice were killed at POD 21, and CFUs were quantified after culture. Myeloperoxidase (MPO) and β-N-acetylglucosaminidase (NAG) were assayed to look at neutrophil infiltration and activated tissue macrophage recruitment, respectively., Results: Serum values confirmed 25D3 deficiency and repletion of the 25D3-rescued group. Bacterial bioluminescence and neutrophil fluorescence were significantly greater (p < 0.05) in the 25D3-deficient group. CFU counts from the joint tissue and implant were also significantly greater in this group (p < 0.05). Rescue treatment significantly decreased bacterial burden and neutrophil infiltration (p < 0.05). Compared with the 25D3-sufficient and 25D3-rescued groups, MPO activity was higher (p < 0.02) and NAG activity was lower (p < 0.03) in the 25D3-deficient group., Conclusions: This study demonstrated in vivo in a mouse model of PJI that (1) 25D3 deficiency results in increased bacterial burden and neutrophil infiltration, and (2) this effect can be reversed with preoperative repletion of 25D3., Clinical Relevance: Considering that >65% of patients undergoing arthroplasty have insufficient or low levels of total 25D and that 25D levels can be replenished with ease using a U.S. Food and Drug Administration (FDA)-approved, oral 25D3 product, 25D deficiency may be an important modifiable risk factor in humans undergoing joint replacement.

Context: Vitamin D deficiency disproportionately affects nonwhite individuals. Controversy persists over how to best restore low 25D levels, and how to best define vitamin D status [total (protein bound plus free) vs free 25D]., Objective: To assess the effects of vitamin D3 (cholecalciferol, or D3) vs 25-hydroxyvitamin D3 (calcifediol, or 25D3) on total and free 25D in a multiethnic cohort of adults, and whether change in parathyroid hormone (PTH) is more strongly associated with total vs free 25D., Design: Sixteen-week randomized controlled trial. Biochemistries at 0, 4, 8, and 16 weeks., Setting: Academic medical center., Participants: Thirty-five adults ≥18 years of age with 25D levels <20 ng/mL., Intervention: Sixty micrograms (2400 IU)/d of D3 or 20 μg/d of 25D3., Main Outcome Measures: Total and free 25D, and PTH., Results: Baseline total (16.2 ± 3.7 vs 17.0 ± 2.5 ng/mL; P = 0.4) and free (4.2 ± 0.8 vs 4.7 ± 1.0 pg/mL; P = 0.2) 25D were similar between D3 and 25D3 groups, respectively; 25D3 increased total (+25.5 vs +13.8 ng/mL; P = 0.001) and free (+6.6 vs +3.5 pg/mL; P = 0.03) 25D more than D3. By 4 weeks, 87.5% of 25D3 participants had total 25D levels ≥30 ng/mL, compared with 23.1% of D3 participants (P = 0.001). Change in PTH was associated with both total (P = 0.01) and free 25D (P = 0.04)., Conclusions: 25D3 increased total and free 25D levels more rapidly than D3, regardless of race/ethnicity. Free and total 25D were similarly associated with change in PTH., (Copyright © 2017 Endocrine Society)

Introduction: Epidemiology has linked preeclampsia (PET) to decreased maternal serum 25-hydroxyvitamin D3 (25(OH)D3). However, alterations in systemic and placental/decidual transport and metabolism of 25(OH)D3 during pregnancy suggest that other forms of vitamin D may also contribute to the pathophysiology of PET., Methods: In a cross sectional analysis of normal pregnant women at 1st (n = 25) and 3rd trimester (n = 21), pregnant women with PET (n = 22), and non-pregnant female controls (n = 20) vitamin D metabolites were quantified in paired maternal serum, placental, and decidual tissue., Results: Serum 25(OH)D3 was not significantly different in sera across all four groups. In normal 3rd trimester pregnant women serum active 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D3) was significantly higher than non-pregnant, normal 1st trimester pregnant, and PET women. Conversely, PET sera showed highest levels of the catabolites 3-epi-25(OH)D3 and 24,25-dihydroxyvitamin D3 (24,25(OH) 2 D3). Serum albumin was significantly lower in normal 3rd trimester pregnant women and PET relative to normal 1st trimester pregnant women, but there was no change in free/bioavailable 25(OH)D3. In PET placental tissue, 25(OH)D3 and 3-epi-25(OH)D3 were lower than normal 3rd trimester tissue, whilst placental 24,25(OH) 2 D3 was highest in PET. Tissue 1,25(OH) 2 D3 was detectable in 1st trimester decidua, which also showed 10-fold higher 25(OH)D3 relative to paired placentae. 3-epi-25(OH)D3 and 24,25(OH) 2 D3 were not different for decidua and placenta. In normal 3rd trimester pregnant women, total, free and bioavailable maternal 25(OH)D3 correlated with placental 25(OH)D3, but this was not conserved for PET., Discussion: These data indicate that PET is associated with decreased activation, increased catabolism, and impaired placental uptake of 25(OH)D3., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) 2 D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH) 2 D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH) 2 D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH) 2 D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH) 2 D-mediated induction of transcription., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

25-Hydroxyvitamin D (25D) circulates bound primarily to serum vitamin D binding protein (DBP), with DBP showing higher binding affinity for 25D3 than 25D2. We therefore hypothesized that vitamin D2 (D2) promotes higher serum levels of unbound 25D (free 25D), with different functional responses, relative to vitamin D3 (D3). Week 3 C56BL/6 mice were placed on diets containing either D2 or D3 alone (both 1000 IU/kg). At week 8 and week 16, D2 mice had only 25D2 in circulation (26.6 ± 1.9 and 33.3 ± 4.4 ng/mL), and D3 mice had only 25D3 (28.3 ± 2.0 and 31.7 ± 2.1 ng/mL). At week 8 (44.5 ± 6.4 vs 62.4 ± 11.6 pg/mL, P < .05) and week 16 (78.4 ± 12.6 vs 95.5 ± 11.6), D2 mice had lower serum 1,25-dihydroxyvitamin D relative to D3 mice. By contrast, measured free 25D was significantly higher in D2 mice at week 8 (16.8 ± 0.65 vs 8.4 ± 0.63 pg/mL, P < .001) and week 16 (17.4 ± 0.43 vs 8.4 ± 0.44, P < .001). A two-way ANOVA of bone histomorphometry showed that week 8 D2 mice had significantly higher osteoclast surface/bone surface, eroded surface/bone surface, and mineral apposition rate compared with D3 mice. Osteoblast surface/bone surface was higher in week 8 D2 females but not week 8 D2 males. At week 16, D2 mice had significantly higher bone volume/total volume and trabecular number compared with D3 mice. Differences in bone phenotype were observed despite D2 mice reaching similar serum 25D levels and lower 1,25D levels compared with D3 mice. These data indicate that 25D2 binds less well to DBP than 25D3, with resulting higher levels of free 25D promoting differential effects on bone in mice exposed to D2 alone.

Context: Controversy persists over: 1) how best to restore low serum 25-hydroxyvitamin D (25D) levels (vitamin D2 [D2] vs vitamin D3 [D3]); 2) how best to define vitamin D status (total [protein-bound + free] vs free 25D); and 3) how best to assess the bioactivity of free 25D., Objective: To assess: 1) the effects of D2 vs D3 on serum total and free 25D; and 2) whether change in intact PTH (iPTH) is more strongly associated with change in total vs free 25D., Design: Participants previously enrolled in a D2 vs D3 trial were matched for age, body mass index, and race/ethnicity. Participants received 50 000 IU of D2 or D3 twice weekly for 5 weeks, followed by a 5-week equilibration period. Biochemical assessment was performed at baseline and at 10 weeks., Setting and Participants: Thirty-eight adults (19 D2 and 19 D3) ≥18 years of age with baseline 25D levels <30 ng/mL were recruited from an academic ambulatory osteoporosis clinic., Outcome Measures: Serum measures were total 25D, free 25D (directly measured), 1,25-dihydroxyvitamin D, calcium, and iPTH. Urine measure was fasting calcium:creatinine ratio., Results: Baseline total (22.2 ± 3.3 vs 23.3 ± 7.2 ng/mL; P = .5) and free (5.4 ± 0.8 vs 5.3 ± 1.7 pg/mL; P = .8) 25D levels were similar between D2 and D3 groups. Increases in total (+27.6 vs +12.2 ng/mL; P = .001) and free (+3.6 vs +6.2 pg/mL; P = .02) 25D levels were greater with D3 vs D2. Percentage change in iPTH was significantly associated with change in free (but not total) 25D, without and with adjustment for supplementation regimen, change in 1,25-dihydroxyvitamin D, and change in calcium., Conclusions: D3 increased total and free 25D levels to a greater extent than D2. Free 25D may be superior to total 25D as a marker of vitamin D bioactivity.

Context: Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitamin D binding protein (DBP) used to calculate f25OHD., Objectives: Our objective was to assess racial-geographic differences in f25OHD and to understand inconsistencies in racial associations with DBP and calculated f25OHD., Design: This study used a cross-sectional design., Setting: The general community in the United States, United Kingdom, and The Gambia were included in this study., Participants: Men in Osteoporotic Fractures in Men and Medical Research Council studies (N = 1057) were included., Exposures: Total 25OHD concentration, race, and DBP (GC) genotype exposures were included., Outcome Measures: Directly measured f25OHD, DBP assessed by proteomics, monoclonal and polyclonal immunoassays, and calculated f25OHD were the outcome measures., Results: Total 25OHD correlated strongly with directly measured f25OHD (Spearman r = 0.84). Measured by monoclonal assay, mean DBP in African-ancestry subjects was approximately 50% lower than in whites, whereas DBP measured by polyclonal DBP antibodies or proteomic methods was not lower in African-ancestry. Calculated f25OHD (using polyclonal DBP assays) correlated strongly with directly measured f25OHD (r = 0.80-0.83). Free 25OHD, measured or calculated from polyclonal DBP assays, reflected total 25OHD concentration irrespective of race and was lower in African Americans than in US whites., Conclusions: Previously reported racial differences in DBP concentration are likely from monoclonal assay bias, as there was no racial difference in DBP concentration by other methods. This confirms the poor vitamin D status of many African-Americans and the utility of total 25OHD in assessing vitamin D in the general population.

Rationale: Vitamin D deficiency, often defined by total serum 25-hydroxyvitamin D (25[OH]D) <20 ng/ml, is common in critically ill patients, with associations with increased mortality and morbidity in the intensive care unit. Correction of vitamin D deficiency in critical illness has been recommended, and ongoing clinical trials are investigating the effect of repletion on patient outcome. The biologically active amount of 25(OH)D depends on the concentration and protein isoform of vitamin D-binding protein (VDBP), which is also an acute-phase reactant affected by inflammation and injury., Objectives: We performed a secondary analysis of a cohort of critically ill children in which we reported a high rate of vitamin D deficiency, to examine how VDBP level and genotype would impact vitamin D status., Methods: We prospectively enrolled 511 children admitted to the pediatric intensive care unit over a 12-month period., Measurements and Main Results: We measured serum VDBP in 479 children. We genotyped single nucleotide polymorphisms rs7041 and rs4588 in the VDBP gene (GC) to determine haplotypes GC1F, GC1S, and GC2 in 178 subjects who consented, then calculated bioavailable 25(OH)D from serum 25(OH)D, VDBP, albumin, and GC haplotype. The median serum VDBP level was 159 μg/ml (interquartile range, 108-221), lower than has been reported in healthy children. Factors predicting lower levels in multivariate analysis included age <1 year, nonwhite race, being previously healthy, 25(OH)D <20 ng/ml and greater illness severity. In the subgroup that was genotyped, GC haplotype had the strongest association with VDBP level; carriage of one additional copy of GC1S was associated with a 37.5% higher level (95% confidence interval, 31.9-44.8; P < 0.001). Bioavailable 25(OH)D was also inversely associated with illness severity (r = -0.24, P < 0.001), and ratio to measured total 25(OH)D was variable and related to haplotype., Conclusions: Physiologic deficiency of 25(OH)D in critical illness may be more difficult to diagnose, given that lower VDBP levels increase bioavailability. Treatment studies conducted on the basis of total 25(OH)D level, without consideration of VDBP concentration and genotype, may increase the risk of falsely negative results.

The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Human monocytes activated by toll-like receptor 2/1 ligand (TLR2/1L) show enhanced expression of the vitamin D receptor (VDR) and the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). The resulting intracrine conversion of precursor 25-hydroxyvitamin D3 (25OHD) to active 1,25-dihydroxyvitamin D (1,25(OH)2D) can stimulate expression of antibacterial cathelicidin (CAMP). To determine whether this response is functional in HIV-infected subjects (HIV+ ), serum from HIV+ subjects pre- and post-vitamin D supplementation was utilized in monocyte cultures with or without TLR2/1L. Expression of CYP27B1 and VDR was enhanced following treatment with TLR2/1L, although this effect was lower in HIV+ vs HIV- serum (p<0.05). CAMP was also lower in TLR2/1L-treated monocytes cultured in HIV+ serum (p<0.01). In a dose study, supplementation of HIV+ subjects with 4000IU or 7000IU vitamin D/day increased serum 25OHD from 17.3±8.0 and 20.6±6.2ng/ml (43nM and 51nM) at baseline to 41.1±12.0 and 51.9±23.1ng/ml (103nM and 130nM) after 12 weeks (both p<0.001). Greater percent change from baseline 25OHD was significantly associated with enhanced TLR2/1L-induced monocyte CAMP adjusted for baseline expression (p=0.009). In a randomized placebo-controlled trial, 7000IU vitamin D/day increased serum 25OHD from 18.0±8.6 to 32.7±13.8ng/ml (45nM and 82nM) after 12 weeks. Expression of CAMP increased significantly from baseline after 52 weeks of vitamin D-supplementation. At this time point, TLR2/1L-induced CAMP was positively associated with percent change from baseline in 25OHD (p=0.029 overall and 0.002 within vitamin D-supplemented only). These data indicate that vitamin D supplementation in HIV-infected subjects can promote improved antibacterial immunity, but also suggest that longer periods of supplementation are required to achieve this., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Patients with chronic kidney disease (CKD), who usually display low serum 25-hydroxyvitamin D (25D) and 1,25-dihydroxyvitamin D (1,25D), are at high risk of infection, notably those undergoing peritoneal dialysis (PD). We hypothesized that peritoneal macrophages from PD patients are an important target for vitamin D-induced antibacterial activity. Dialysate effluent fluid was obtained from 27 non-infected PD patients. Flow cytometry indicated that PD cells were mainly monocytic (37.9±17.7% cells CD14+/CD45+). Ex vivo analyses showed that PD cells treated with 25D (100 nM, 6 hrs) or 1,25D (5 nM, 6 hrs) induced mRNA for antibacterial cathelicidin (CAMP) but conversely suppressed mRNA for hepcidin (HAMP). PD cells from patients with peritonitis (n = 3) showed higher baseline expression of CAMP (18-fold±9, p<0.05) and HAMP (64-fold±7) relative to cells from non-infected patients. In 12 non-infected PD patients, oral supplementation with a single dose of vitamin D2 (100,000 IU) increased serum levels of 25D from 18±8 to 41±15 ng/ml (p = 0.002). This had no significant effect on PD cell CD14/CD45 expression, but mRNA for HAMP was suppressed significantly (0.5-fold, p = 0.04). Adjustment for PD cell CD14/CD45 expression using a mixed linear statistical model also revealed increased expression of CAMP (mRNA in PD cells and protein in effluent) in vitamin D-supplemented patients. These data show for the first time that vitamin D supplementation in vitro and in vivo promotes innate immune responses that may enhance macrophage antibacterial responses in patients undergoing PD. This highlights a potentially important function for vitamin D in preventing infection-related complications in CKD.

Activation of precursor 25-hydroxyvitamin D3 (25D) to hormonal 1,25-dihydroxyvitamin D3 (1,25D) is a pivotal step in vitamin D physiology, catalysed by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). To establish new models for assessing the physiological importance of the 1α-hydroxylase-25D-axis, we used Danio rerio (zebrafish) to characterize expression and biological activity of the gene for 1α-hydroxylase (cyp27b1). Treatment of day 5 zebrafish larvae with inactive 25D (5-150 nM) or active 1,25D (0.1-10 nM) induced dose responsive expression (15-95-fold) of the vitamin D-target gene cyp24a1 relative to larvae treated with vehicle, suggesting the presence of Cyp27b1 activity. A full-length zebrafish cyp27b1 cDNA was then generated using RACE and RT-PCR methods. Sequencing of the resulting clone revealed an open reading frame encoding a protein of 505 amino acids with 54% identity to human CYP27B1. Transfection of a cyp27b1 expression vector into HKC-8, a human kidney proximal tubular epithelial cell line, enhanced intracrine metabolism of 25D to 1,25D resulting in greater than twofold induction of CYP24A1 mRNA expression and a 25-fold increase in 1,25D production compared to empty vector. These data indicate that we have cloned a functional zebrafish CYP27B1, representing a phylogenetically distant branch from mammals of this key enzyme in vitamin D metabolism. Further analysis of cyp27b1 expression and activity in zebrafish may provide new perspectives on the biological importance of 25D metabolism., (Copyright © 2014 John Wiley & Sons, Ltd.)

The last five years have witnessed a remarkable renaissance in vitamin D research and a complete re-evaluation of its benefits to human health. Two key factors have catalyzed these changes. First, it now seems likely that localized, tissue-specific, conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)2D) drives many of the newly recognized effects of vitamin D on human health. The second key factor concerns the ongoing discussion as to what constitutes adequate or optimal serum vitamin D (25OHD) status, with the possibility that vitamin D-deficiency is common to communities across the globe. These two concepts appear to be directly linked when low serum concentrations of 25OHD compromise intracrine generation of 1,25(OH)2D within target tissues. But, is this an over-simplification? Pro-hormone 25OHD is a lipophilic molecule that is transported in the circulation bound primarily to vitamin D binding protein (DBP). While the association between 25OHD and DBP is pivotal for renal handling of 25OHD and endocrine synthesis of 1,25(OH)2D, what is the role of DBP for extra-renal synthesis of 1,25(OH)2D? We hypothesize that binding to DBP impairs delivery of 25OHD to the vitamin D-activating enzyme 1α-hydroxylase in some target cells. Specifically, it is unbound, 'free' 25OHD that drives many of the non-classical actions of vitamin D. Levels of 'free' 25OHD are dependent on the concentration of DBP and alternative serum binding proteins such as albumin, but will also be influenced by variations in DBP binding affinity for specific vitamin D metabolites. The aim of this review will be to discuss the merits of 'free 25OHD' as an alternative marker of vitamin D status, particularly in the context of non-classical responses to vitamin D. This article is part of a Special Issue entitled '16th Vitamin D Workshop'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Immunomodulatory responses to the active form of vitamin D (1,25-dihydroxyvitamin D, 1,25D) have been recognized for many years, but it is only in the last 5 years that the potential role of this in normal human immune function has been recognized. Genome-wide analyses have played a pivotal role in redefining our perspective on vitamin D and immunity. The description of increased vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1) expression in macrophages following a pathogen challenge, has underlined the importance of intracrine vitamin D as key mediator of innate immune function. It is now clear that both macrophages and dendritic cells (DCs) are able to respond to 25-hydroxyvitamin D (25D), the major circulating vitamin D metabolite, thereby providing a link between the function of these cells and the variations in vitamin D status common to many humans. The identification of hundreds of primary 1,25D target genes in immune cells has also provided new insight into the role of vitamin D in the adaptive immune system, such as the modulation of antigen-presentation and T cells proliferation and phenotype, with the over-arching effects being to suppress inflammation and promote immune tolerance. In macrophages 1,25D promotes antimicrobial responses through the induction of antibacterial proteins, and stimulation of autophagy and autophagosome activity. In this way variations in 25D levels have the potential to influence both innate and adaptive immune responses. More recent genome-wide analyses have highlighted how cytokine signaling pathways can influence the intracrine vitamin D system and either enhance or abrogate responses to 25D. The current review will discuss the impact of intracrine vitamin D metabolism on both innate and adaptive immunity, whilst introducing the concept of disease-specific corruption of vitamin D metabolism and how this may alter the requirements for vitamin D in maintaining a healthy immune system in humans.

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D2) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing. More recently hnRNP C has also been shown to function as a DNA binding protein exerting a dominant-negative effect on transcriptional responses to the vitamin D hormone,1,25-dihydroxyvitamin D (1,25(OH) 2 D), via interaction in cis with vitamin D response elements (VDREs). The physiologically active form of human hnRNPC is a tetramer of hnRNPC1 (huC1) and C2 (huC2) subunits known to be critical for specific RNA binding activity in vivo , yet the requirement for heterodimerization of huC1 and C2 in DNA binding and downstream action is not well understood. While over-expression of either huC1 or huC2 alone in mouse osteoblastic cells did not suppress 1,25(OH) 2 D-induced transcription, over-expression of huC1 and huC2 in combination using a bone-specific polycistronic vector successfully suppressed 1,25(OH) 2 D-mediated induction of osteoblast target gene expression. Over-expression of either huC1 or huC2 in human osteoblasts was sufficient to confer suppression of 1,25(OH) 2 D-mediated transcription, indicating the ability of transfected huC1 and huC2 to successfully engage as heterodimerization partners with endogenously expressed huC1 and huC2. The failure of the chimeric combination of mouse and human hnRNPCs to impair 1,25(OH) 2 D-driven gene expression in mouse cells was structurally predicted, owing to the absence of the last helix in the leucine zipper (LZ) heterodimerization domain of hnRNPC gene product in lower species, including the mouse. These results confirm that species-specific heterodimerization of hnRNPC1 and hnRNPC2 is a necessary prerequisite for DNA binding and down-regulation of 1,25(OH) 2 D-VDR-VDRE-directed gene transactivation in osteoblasts.

When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms., (Copyright © 2013 American Society for Bone and Mineral Research.)

Vitamin D is a potent stimulator of monocyte innate immunity, and this effect is mediated via intracrine conversion of 25-hydroxyvitamin D (25OHD) to 1,25-dihydroxyvitamin D (1,25(OH)(2) D). In the kidney, synthesis of 1,25(OH)(2) D is suppressed by fibroblast growth factor 23 (FGF23), via transcriptional suppression of the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). We hypothesized that FGF23 also suppresses CYP27B1 in monocytes, with concomitant effects on intracrine responses to 1,25(OH)(2) D. Healthy donor peripheral blood mononuclear cell monocytes (PBMCm) and peritoneal dialysate monocyte (PDm) effluent from kidney disease patients were assessed at baseline to confirm the presence of mRNA for FGF23 receptors (FGFRs), with Klotho and FGFR1 being more strongly expressed than FGFR2/3/4 in both cell types. Immunohistochemistry showed coexpression of Klotho and FGFR1 in PBMCm and PDm, with this effect being enhanced following treatment with FGF23 in PBMCm but not PDm. Treatment with FGF23 activated mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) pathways in PBMCm, demonstrating functional FGFR signaling in these cells. FGF23 treatment of PBMCm and PDm decreased expression of mRNA for CYP27B1. In PBMCm this was associated with downregulation of 25OHD to 1,25(OH)(2) D metabolism, and concomitant suppression of intracrine induced 24-hydroxylase (CYP24A1) and antibacterial cathelicidin (LL37). FGF23 suppression of CYP27B1 was particularly pronounced in PBMCm treated with interleukin-15 to stimulate synthesis of 1,25(OH)(2) D. These data indicate that FGF23 can inhibit extra-renal expression of CYP27B1 and subsequent intracrine responses to 1,25(OH)(2) D in two different human monocyte models. Elevated expression of FGF23 may therefore play a crucial role in defining immune responses to vitamin D and this, in turn, may be a key determinant of infection in patients with chronic kidney disease (CKD)., (Copyright © 2013 American Society for Bone and Mineral Research.)

The serum vitamin D binding protein (DBP), also known as GC-globulin, is a multifunctional protein known for its role in the transport of vitamin D metabolites. DBP also binds fatty acids and actin monomers, preventing their polymerization that could be detrimental in the circulatory system. DBP may have immune functions independent of its role as a transporter of vitamin D. Because of the abundance of DBP, many aspects of its basic biochemistry were quickly established. Other features of vitamin D action, particularly transcriptional mechanisms of regulation, received greater focus and early interest in DBP centred on its value as a tool for population genetics because of its intriguing genetic variations. Nonetheless, knowledge of DBP mechanisms in physiology was obtained, and functions beyond vitamin D ligand binding were identified. With the recent increased attention regarding the benefits of vitamin D (bone health and immunological regulation), there has been a resurgence of interest in DBP. Because DBP is the primary transporter of vitamin D, it has a role in maintaining the total levels of vitamin D for the organism and in regulating the amounts of free (unbound) vitamin D available for specific tissues and cell types to utilize. This review will describe the findings on the basic biochemistry and molecular biology of DBP, the studies that elucidated its biological functions and highlight these results in light of the current renewed interest in vitamin D and human health, as well as the debate over what constitutes sufficient levels of vitamin D., (Copyright © 2012 John Wiley & Sons, Ltd.)

Vitamin D binding protein (DBP) plays a key role in the bioavailability of active 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and its precursor 25-hydroxyvitamin D (25OHD), but accurate analysis of DBP-bound and free 25OHD and 1,25(OH)(2)D is difficult. To address this, two new mathematical models were developed to estimate: 1) serum levels of free 25OHD/1,25(OH)(2)D based on DBP concentration and genotype; 2) the impact of DBP on the biological activity of 25OHD/1,25(OH)(2)D in vivo. The initial extracellular steady state (eSS) model predicted that 50 nM 25OHD and 100 pM 1,25(OH)(2)D), <0.1% 25OHD and <1.5% 1,25(OH)(2)D are 'free' in vivo. However, for any given concentration of total 25OHD, levels of free 25OHD are higher for low affinity versus high affinity forms of DBP. The eSS model was then combined with an intracellular (iSS) model that incorporated conversion of 25OHD to 1,25(OH)(2)D via the enzyme CYP27B1, as well as binding of 1,25(OH)(2)D to the vitamin D receptor (VDR). The iSS model was optimized to 25OHD/1,25(OH)(2)D-mediated in vitro dose-responsive induction of the vitamin D target gene cathelicidin (CAMP) in human monocytes. The iSS model was then used to predict vitamin D activity in vivo (100% serum). The predicted induction of CAMP in vivo was minimal at basal settings but increased with enhanced expression of VDR (5-fold) and CYP27B1 (10-fold). Consistent with the eSS model, the iSS model predicted stronger responses to 25OHD for low affinity forms of DBP. Finally, the iSS model was used to compare the efficiency of endogenously synthesized versus exogenously added 1,25(OH)(2)D. Data strongly support the endogenous model as the most viable mode for CAMP induction by vitamin D in vivo. These novel mathematical models underline the importance of DBP as a determinant of vitamin D 'status' in vivo, with future implications for clinical studies of vitamin D status and supplementation.

TB remains a major cause of mortality throughout the world. Low vitamin D status has been linked to increased risk of TB and other immune disorders. These observations suggest a role for vitamin D as a modulator of normal human immune function. This article will detail the cellular and molecular mechanisms by which vitamin D regulates the immune system and how vitamin D insufficiency may lead to immune dysregulation. The importance of vitamin D bioavailability as a mechanism for defining the immunomodulatory actions of vitamin D and its impact on TB will also be discussed. The overall aim will be to provide a fresh perspective on the potential benefits of vitamin D supplementation in the prevention and treatment of TB.

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background: Serum 25-hydroxyvitamin D (25OHD) is a key factor in determining monocyte induction of the antimicrobial protein cathelicidin, which requires intracrine conversion of 25OHD to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. Both vitamin D metabolites circulate bound to vitamin D-binding protein (DBP), but the effect of this on induction of monocyte cathelicidin remains unclear., Methods: Human monocytes were cultured in medium containing 1) serum from DBP knockout (DBP(-/-)) or DBP(+/-) mice, 2) serum-free defined supplement reconstituted with DBP or albumin (control), and 3) human serum with different DBP [group-specific component [Gc]] genotypes with varying affinities for vitamin D metabolites. In each case, response to added 1,25(OH)(2)D(3) or 25OHD(3) was determined by measuring expression of mRNA for cathelicidin and 24-hydroxylase. Monocyte internalization of DBP was assessed by fluorescent tagging followed by microscopic and flow cytometric analysis of tagged DBP., Results: Monocytes cultured in DBP(-/-) serum showed more potent induction of cathelicidin by 25OHD(3) or 1,25(OH)(2)D(3) when compared with DBP(+/-) serum. Likewise, DBP added to serum-free medium attenuated 25OHD(3)/1,25(OH)(2)D(3) responses. Fluorescently tagged DBP showed low-level uptake by monocytes, but this did not appear to involve a megalin-mediated mechanism. Human serum containing low-affinity Gc2-1S or Gc2-2, respectively, supported 2.75-fold (P = 0.003) and 2.43-fold (P = 0.016) higher induction of cathelicidin by 25OHD relative to cells cultured with high affinity Gc1F-1F., Conclusion: These data indicate that DBP plays a pivotal role in regulating the bioavailablity of 25OHD to monocytes. Vitamin D-dependent antimicrobial responses are therefore likely to be strongly influenced by DBP polymorphisms.

Vitamin D insufficiency is a global health issue. Although classically associated with rickets, low vitamin D levels have also been linked to aberrant immune function and associated health problems such as inflammatory bowel disease (IBD). To test the hypothesis that impaired vitamin D status predisposes to IBD, 8-wk-old C57BL/6 mice were raised from weaning on vitamin D-deficient or vitamin D-sufficient diets and then treated with dextran sodium sulphate (DSS) to induce colitis. Vitamin D-deficient mice showed decreased serum levels of precursor 25-hydroxyvitamin D(3) (2.5 +/- 0.1 vs. 24.4 +/- 1.8 ng/ml) and active 1,25-dihydroxyvitamin D(3) (28.8 +/- 3.1 vs. 45.6 +/- 4.2 pg/ml), greater DSS-induced weight loss (9 vs. 5%), increased colitis (4.71 +/- 0.85 vs. 1.57 +/- 0.18), and splenomegaly relative to mice on vitamin D-sufficient chow. DNA array analysis of colon tissue (n = 4 mice) identified 27 genes consistently (P < 0.05) up-regulated or down-regulated more than 2-fold in vitamin D-deficient vs. vitamin D-sufficient mice, in the absence of DSS-induced colitis. This included angiogenin-4, an antimicrobial protein involved in host containment of enteric bacteria. Immunohistochemistry confirmed that colonic angiogenin-4 protein was significantly decreased in vitamin D-deficient mice even in the absence of colitis. Moreover, the same animals showed elevated levels (50-fold) of bacteria in colonic tissue. These data show for the first time that simple vitamin D deficiency predisposes mice to colitis via dysregulated colonic antimicrobial activity and impaired homeostasis of enteric bacteria. This may be a pivotal mechanism linking vitamin D status with IBD in humans.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, [1,25(OH)2D3] has potent actions on innate and adaptive immunity. Although endocrine synthesis of 1,25(OH)2D3 takes place in the kidney, the enzyme that catalyzes this, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27b1 in humans, Cyp27b1 in mice), is expressed at many extra-renal sites including the colon. We have shown previously that colonic expression of CYP27b1 may act to protect against the onset of colitis. To investigate this further, we firstly characterized changes in Cyp27b1 expression in a mouse model of colitis. Mice treated with dextran sodium sulfate (DSS) showed weight loss, histological evidence of colitis, and increased expression of inflammatory cytokines. This was associated with decreased renal expression of Cyp27b1 (5-fold, P=0.013) and lower serum 1,25(OH)2D3 (51.8+/-5.9 pg/nl vs. 65.1+/-1.6 in controls, P<0.001). However, expression of CYP27b1 was increased in the proximal colon of DSS mice (4-fold compared with controls, P<0.001). Further studies were carried out using Cyp27b1 null (-/-) mice. Compared with+/-controls the Cyp27b1-/-mice showed increased weight loss (4.9% vs. 22.8%, P<0.001) and colitis. This was associated with raised IL-1 in the distal colon and IL-17 in the proximal and distal colon. Conversely, DSS-treated Cyp27b1-/-mice exhibited lower IL-10 in the proximal colon and toll-like receptors 2 and 4 in the distal colon. These data indicate that both local and endocrine synthesis of 1,25(OH)2D3 affect colitis in DSS-treated mice. Lack of Cyp27b1 exacerbates disease in this model, suggesting that similar effects may occur with vitamin D deficiency.

Our perception of the vitamin D system continues to evolve. Recent studies have re-evaluated the parameters for adequate vitamin D status in humans, revealing a high prevalence of insufficiency in many populations throughout the world. Other reports have highlighted the potential consequences of vitamin D insufficiency beyond established effects on bone homeostasis. Most notably, there is now strong evidence of a role for vitamin D in modulating innate and adaptive immunities, with insufficiency being linked to infectious disease and other immune disorders. To date, signaling pathways for these new responses to vitamin D have been based on established endocrine models for active 1,25-dihydroxyvitamin D, despite present evidence for more localized, intracrine modes of action. In the following review, we provide a fresh perspective on vitamin D signaling in non-classical target cells such as macrophages by highlighting novel factors associated with the transport and action of this pluripotent secosteroid.

The constitutively expressed member of the heat shock protein-70 family (hsc70) is a chaperone with multiple functions in cellular homeostasis. Previously, we demonstrated the ability of hsc70 to bind 25-hydroxyvitamin D3 (25-OHD3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Hsc70 also recruits and interacts with the co-chaperone Bcl2-associated athanogene (BAG)-1 via the ATP-binding domain that resides on hsc70. Competitive ligand-binding assays showed that, like hsc70, recombinant BAG-1 is able to bind 25-OHD3 (Kd=0.71+/-0.25 nM, Bmax 69.9+/-16.1 fmoles/microg protein) and 1,25(OH)2D3 (Kd=0.16+/-0.07 nM, Bmax = 38.1+/-3.5 fmoles/microg protein; both n=3 separate binding assays, P<0.001 for Kd and Bmax). To investigate the functional significance of this, we transiently overexpressed the S, M, and L variants of BAG-1 into human kidney HKC-8 cells stably transfected with a 1,25(OH)2D3-responsive 24-hydroxylase (CYP24) promoter-reporter construct. As HKC-8 cells also express the enzyme 1alpha-hydroxylase, both 25-OHD3 (200 nM) and 1,25(OH)2D3 (5 nM) were able to induce CYP24 promoter activity. This was further enhanced following overexpression of all the three BAG-1 isoforms. By contrast, BAG-1 isoforms had no effect on metabolism of 25-OHD3 by HKC-8 cells (either via 1alpha- or 24-hydroxylase activities). Further studies showed that a mutant form of BAG-1S exhibited decreased binding of 1,25(OH)2D3 and this resulted in a concomitant loss of potentiation of CYP24 promoter transactivation. Similar effects were not observed for 25-OHD3. These data highlight a novel role for BAG-1 as an intracellular-binding protein for 1,25(OH)2D3 and further suggest that BAG-1 is able to potentiate vitamin D receptor-mediated transactivation by acting as a nuclear chaperone for 1,25(OH)2D3.

We undertook an investigation of an outbreak of rachitic bone disease in the Emperor Tamarin New World primate colony at the Los Angeles Zoo in the mid-1980s. The disease phenotype resembled that observed in humans with an inactivating mutation of the vitamin D receptor (VDR), hypocalcemia, high 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) levels, and rickets in rapidly growing adolescent primates. In contrast to the human disease, the New World primate VDR was functionally normal in all respects. The proximate cause of vitamin D hormone resistance in New World primates was determined to be the constitutive overexpression of a heterogeneous nuclear ribonucleoprotein in the A family which we coined the vitamin D response element binding protein (VDRE-BP). VDRE-BP competed in trans with the VDR-retinoid X receptor (RXR) for binding to the vitamin D response element. VDRE-BP-legislated resistance to 1,25-(OH)(2)D was antagonized (i.e., compensated) by another set of constitutively overexpressed proteins, the hsp-70-related intracellular vitamin D binding proteins (IDBPs). IDBPs, present but expressed at much lower levels in Old World primates including man, exhibited a high capacity for 25-hydroxylated vitamin D metabolites and functioned to traffic vitamin Ds to specific intracellular destinations to promote their action and metabolism., (Copyright 2002 Wiley-Liss, Inc.)

New World primates (NWPs) have high circulating 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels. Comparable levels would be harmful to Old World primates (OWPs) and humans. Thus, NWPs must have developed mechanisms of 1,25-(OH)2D resistance to survive. In humans, patients with hypocalcemic vitamin D-resistant rickets type II have high circulating vitamin D levels and vitamin D resistance due to expression of a dysfunctional vitamin D receptor (VDR). To examine if this could wholly or in part explain vitamin D resistance in NWPs, VDR from Saguinus oedipus (cotton top tamarin) NWP B95-8 cells was cloned by reverse-transcription polymerase chain reaction (RT-PCR). The NWP VDR cDNA sequence showed 96% homology at the DNA level and 98% homology at the amino acid level compared to human VDR. To assay for function, NWP VDR cDNA was transiently transfected into CV-1 cells with a vitamin D response element reporter plasmid. No difference between OWP and NWP VDR-directed transactivation was observed. These results indicate that the mechanism of vitamin D resistance in NWPs is not due to a dysfunctional VDR, and is consistent with our hypothesis that vitamin D resistance in NWPs is mediated by overexpression of a VDR-independent vitamin D response element binding protein.

Previously recognized intracellular proteins with an affinity for vitamin D metabolites include the vitamin D receptor and the cytochrome P-450-based vitamin D metabolizing mixed-function oxidases. We recently characterized a third set of high-capacity, intracellular vitamin D binding proteins (IDBPs) in the inducible heat shock protein-70 (hsp-70) family. Here we report the cloning and expression of cDNAs coding for two IDBPs. The full-length cDNAs for IDBP-1 and IDBP-2 demonstrated 95% and 94% nucleotide homology, respectively, with the cDNAs for human constitutively expressed heat shock protein 70 (hsc-70) and hsp-70. Transient expression of the IDBP cDNAs in a vitamin D-responsive primate cell line increased extractable 25-hydroxylated vitamin D metabolite-IDBP-binding 25-fold. Transfection experiments also demonstrated that the majority of the constitutively expressed 25-hydroxylated vitamin D metabolite binding activity was attributable to expression of the hsc-70-related IDBP-1 and that metabolite binding activity sublocalized to the highly conserved ATP-binding/ATPase domain of hsp-70s. Stable overexpression of IDBP-1 in wild-type cells enhanced vitamin D-directed responsiveness of endogenous vitamin D-24-hydroxylase, osteopontin, and osteocalcin genes by several-fold over that observed in cells transfected with an empty vector. These results suggest that IDBP-1 facilitates the intracellular localization of active vitamin D metabolites and vitamin D receptor-mediated transactivation.

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), in part through activity of the IFN-inducible protein kinase PKR. To escape this antiviral effect, HIV-1 has developed strategies for blocking PKR function. We have previously shown that the HIV-1 Tat protein can associate with PKR in vitro and in vivo and inhibit PKR activity. Here we present evidence that Tat can inhibit PKR activity by both RNA-dependent and RNA-independent mechanisms. Tat inhibited PKR activation by the non-RNA activator heparin, and also suppressed PKR basal level autophosphorylation in the absence of RNA. However, when Tat and dsRNA were preincubated, the amount of Tat required to inhibit PKR activation by dsRNA depended on the dsRNA concentration. In addition to its function in vitro, Tat can also reverse translation inhibition mediated by PKR in COS cells. The Tat amino acid sequence required for interaction with PKR was mapped to residues 40-58, overlapping the hydrophobic core and basic region of HIV-1 Tat. Alignment of amino acid sequences of Tat and eIF-2alpha indicates similarity between the Tat-PKR binding region and the residues around the eIF-2alpha phosphorylation site, suggesting that Tat and eIF-2alpha may bind to the same site on PKR., (Copyright 2000 Academic Press.)

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67: 6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.

Human chemokine receptor 5 (CCR5) functions as a co-receptor for Human immunodeficiency virus (HIV-1) infection. CCR5 is a seven-transmembrane cell surface receptor. Recently, a naturally occurring mutation of CCR5, ccr5Delta32, has been described. A small number of Caucasians are homozygously ccr5Delta32/ccr5Delta32, while a larger number of individuals are heterozygously CCR5/ccr5Delta32. The ccr5Delta32/ccr5Delta32 genotype has been linked to a phenotype that is "highly" protected from HIV-1 infection. On the other hand, several studies have shown that the CCR5/ccr5Delta32 genotype confers "relative" protection from AIDS with onset of disease being delayed by 2-4 years. Although it is known that peripheral blood lymphocytes from heterozygous individuals (CCR5/ccr5Delta32) support ex vivo HIV-1 replication at a reduced level compared with CCR5/CCR5 cells, the molecular basis for this observation is unknown. Here we report on events that post-translationally modify CCR5. We show that CCR5 progresses through the endoplasmic reticulum prior to appearing on the cell surface. Mature CCR5 can be post-translationally modified by phosphorylation and/or co-translationally by multimerization. By contrast, mutant ccr5Delta32, although retaining the capacity for multimerization, was incapable of being phosphorylated. ccr5Delta32 heterocomplexes with CCR5, and this interaction retains CCR5 in the endoplasmic reticulum resulting in reduced cell surface expression. Thus, co-expression in cells of ccr5Delta32 with CCR5 produces a trans-inhibition by the former of ability by the latter to support HIV-1 infection. Taken together, our findings suggest CCR5/ccr5Delta32 heterodimerization as a molecular explanation for the delayed onset of AIDS in CCR5/ccr5Delta32 individuals.

Tumor necrosis factor alpha (TNFalpha) has pleiotropic effects on cellular metabolism. One of the signaling paths from the TNFalpha receptor induces a stress-activated protein kinase cascade. Components within this TNFalpha kinase cascade include mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK), which regulate the activity of c-Jun N-terminal kinase 1 (JNK1). Currently, molecules upstream of MEKK1 that link TNFalpha receptor to downstream kinases are not well understood. Besides TNFalpha, many other stimuli including several oncoproteins can activate JNK1. In most cases, the signaling cascade(s) leading from oncoproteins to JNK1 is poorly elucidated. We report here that the human T-cell lymphotrophic virus, type I (HTLV-I) oncoprotein, Tax, can activate JNK1. We isolated a novel human cell factor, G-protein pathway suppressor 2 (GPS2), by its ability to bind the HTLV-I oncoprotein, and we show that this factor can potently suppress Tax activation of JNK1. In trying to understand the mechanism of GPS2 activity, we found that it also suppressed TNFalpha activation of JNK1 but not TNFalpha activation of p38 kinase nor phorbol activation of extracellular signal-regulated kinase 2. Because GPS2 has minimal effect on MEKK1- or SEK-regulated JNK1 activity, it could act at a point between the TNFalpha receptor and MEKK1 in the initial step(s) of this kinase cascade. Alternatively, it is not excluded that GPS2 could work in a parallel pathway that leads from TNFalpha to JNK1. GPS2 represents a new molecule that could contribute important insights toward how cytokine- and oncoprotein-mediated signal transduction might converge.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

The carboxyl-terminal domain (CTD) of RNA polymerase (RNAP) II contains multiple repeats with a heptapeptide consensus: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. It has been proposed that phosphorylation of this CTD facilitates clearance and elongation of transcription complexes initiated at the promoters. However, not all transcribed promoters require RNAP II with full-length CTD. Furthermore, different activators can promote capably the transcriptional activity of polymerase II mutants deleted in the CTD. Thus, the role of the RNAP II CTD in transcription and in response to activators remains incompletely understood. To study the role of CTD in the regulated transcription of human retroviruses human-T cell lymphotropic virus I and human immunodeficiency virus 1, we used an alpha-amanitin-resistant system developed previously (Gerber, H. P., Hagmann, M., Seipel, K., Georgiev, O., West, M. A., Litingtung, Y., Schaffner, W., and Corden, J. L. (1995) Nature 374, 660-662). We found that transcription directed by the human T-cell lymphotropic virus I activator protein Tax was strongly promoted by CTD-deficient RNA polymerase II. By contrast, the human immunodeficiency virus 1 activator Tat, which is recruited to the promoter by tethering to a nascent leader RNA, requires CTD-containing polymerase II for transcriptional activity. Biochemically, we characterized that Tat associated with a cellular CTD kinase activity, whereas Tax did not. Concordantly, we found that cellular transcription factor Sp1, which can activate CTD-deficient polymerase II with an efficiency similar to Tax, also failed to bind a CTD kinase. Taken together, these observations address mechanistic corollaries between activators with(out) a linked CTD kinase and regulated transcription by RNA polymerase II moieties with(out) a CTD.