Vitamin D and malabsorptive gastrointestinal conditions: A bidirectional relationship?

This paper is one of the outcomes of the 5th International Conference "Controversies in Vitamin D" held in Stresa, Italy from 15 to 18 September 2021 as part of a series of annual meetings which was started in 2017. The scope of these meetings is to discuss controversial issues about vitamin D. Publication of the outcomes of the meeting in international journals allows a wide sharing of the most recent data with the medical and academic community. Vitamin D and malabsorptive gastrointestinal conditions was one of the topics discussed at the meeting and focus of this paper. Participants to the meeting were invited to review available literature on selected issues related to vitamin D and gastrointestinal system and to present their topic to all participants with the aim to initiate a discussion on the main outcomes of which are reported in this document. The presentations were focused on the possible bidirectional relationship between vitamin D and gastrointestinal malabsorptive conditions such as celiac disease, inflammatory bowel diseases (IBDs) and bariatric surgery. In fact, on one hand the impact of these conditions on vitamin D status was examined and on the other hand the possible role of hypovitaminosis D on pathophysiology and clinical course of these conditions was also evaluated. All examined malabsorptive conditions severely impair vitamin D status. Since vitamin D has known positive effects on bone this in turn may contribute to negative skeletal outcomes including reduced bone mineral density, and increased risk of fracture which may be mitigated by vitamin D supplementation. Due to the immune and metabolic extra-skeletal effects there is the possibility that low levels of vitamin D may negatively impact on the underlying gastrointestinal conditions worsening its clinical course or counteracting the effect of treatment. Therefore, vitamin D status assessment and supplementation should be routinely considered in all patients affected by these conditions. This concept is strengthened by the existence of a possible bidirectional relationship through which poor vitamin D status may negatively impact on clinical course of underlying disease. Sufficient elements are available to estimate the desired threshold vitamin D level above which a favourable impact on the skeleton in these conditions may be obtained. On the other hand, ad hoc controlled clinical trials are needed to better define this threshold for obtaining a positive effect of vitamin D supplementation on occurrence and clinical course of malabsorptive gastrointestinal diseases.

Prevalence of osteoporosis in older male veterans receiving hip-containing computed tomography scans: opportunistic use of biomechanical computed tomography analysis (BCT).

Osteoporosis care in men is suboptimal due to low rates of testing and treatment. Applying biomechanical computed tomography (BCT) analysis to existing CT scans, we found a high proportion of men with osteoporosis have never been diagnosed or treated. BCT may improve identification of patients at high risk of fracture. PURPOSE: Osteoporosis care in men is suboptimal due to low rates of DXA testing and treatment. Biomechanical computed tomography analysis (BCT) can be applied "opportunistically" to prior hip-containing CT scans to measure femoral bone strength and hip BMD. METHODS: In this retrospective, cross-sectional study, we used BCT in male veterans with existing CT scans to investigate the prevalence of osteoporosis, defined by hip BMD (T-score ≤ - 2.5) or fragile bone strength (≤ 3500 N). 577 men, age ≥ 65 with abdominal/pelvic CTs performed in 2017-2019, were randomly selected for BCT analysis. Clinical data were collected via electronic health records and used with the femoral neck BMD T-score from BCT to estimate 10-year hip fracture risks by FRAX. RESULTS: Prevalence of osteoporosis by BCT increased with age (13.5% age 65-74; 18.2% age 75-84; 34.3% age ≥ 85), with an estimated overall prevalence of 18.3% for men age ≥ 65. In those with osteoporosis (n = 108/577), only 38.0% (41/108) had a prior DXA and 18.6% (7/108) had received osteoporosis pharmacotherapy. Elevated hip fracture risk by FRAX (≥ 3%) did not fully capture those with fragile bone strength. In a multivariate logistic regression model adjusted for age, BMI, race, and CT location, end stage renal disease (odds ratio 7.4; 95% confidence interval 2.3-23.9), COPD (2.2; 1.2-4.0), and high-dose inhaled corticosteroid use (3.7; 1.2-11.8) were associated with increased odds of having osteoporosis by BCT. CONCLUSION: Opportunistic BCT in male veterans provides an additional avenue to identify patients who are at high risk of fractures.

Vitamin D Regulation of Immune Function.

PURPOSE OF REVIEW: To review the mechanisms by which vitamin D and its metabolites regulate the immune system to facilitate the ability of the body to prevent and/or treat SARS-CoV2 and other respiratory infections and encourage further research into the role that vitamin D supplementation plays in preventing/treating such infections. RECENT FINDINGS: Vitamin D deficiency is associated with an increased risk of SARS-CoV2 and other respiratory infections. Clinical trials in general demonstrate that correction of vitamin D deficiency reduces the risk of hospitalization, ICU admission, and death from SARS-CoV2 infection. The airway epithelium and alveolar macrophages express the enzyme, CYP27B1, that produces the active metabolite of vitamin D, 1,25(OH)2D, and the vitamin D receptor, VDR. Vitamin D and its metabolites promote the innate immune response, which provides the first line of defense against viral and bacterial infections while restricting the adaptive immune response, which if unchecked promotes the inflammatory response leading to the acute respiratory distress syndrome and death. The rationale for treating vitamin D deficiency to reduce the risk of SARS-CoV2 infection and supplementing patients with vitamin D early in the course of SARS-CoV2 infection rests primarily on the ability of vitamin D metabolites to promote an effective immune response to the infection.

Association of Vitamin D Status and COVID-19-Related Hospitalization and Mortality.

BACKGROUND: The relationship between vitamin D status and COVID-19-related clinical outcomes is controversial. Prior studies have been conducted in smaller, single-site, or homogeneous populations limiting adjustments for social determinants of health (race/ethnicity and poverty) common to both vitamin D deficiency and COVID-19 outcomes. OBJECTIVE: To evaluate the dose-response relationship between continuous 25(OH)D and risk for COVID-19-related hospitalization and mortality after adjusting for covariates associated with both vitamin D deficiency and COVID-19 outcomes. DESIGN: Retrospective cohort study. PATIENTS: Veteran patients receiving care in US Department of Veteran Affairs (VA) health care facilities with a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test and a blood 25(OH)D test between February 20, 2020, and November 8, 2020, followed for up to 60 days. MAIN MEASURES: Exposure was blood 25(OH)D concentration ascertained closest to and within 15 to 90 days preceding an index positive SARS-CoV-2 test. Co-primary study outcomes were COVID-19-related inpatient hospitalization requiring airborne, droplet, contact, or other isolation and mortality ascertained within 60 days of an index positive SARS-CoV-2 test. KEY RESULTS: Of 4,599 veterans with a positive SARS-CoV-2 test, vitamin D deficiency (< 20 ng/mL) was identified in 665 (14.5%); 964 (21.0%) were hospitalized; and 340 (7.4%) died. After adjusting for all covariates, including race/ethnicity and poverty, there was a significant independent inverse dose-response relationship between increasing continuous 25(OH)D concentrations (from 15 to 60 ng/mL) and decreasing probability of COVID-19-related hospitalization (from 24.1 to 18.7%, p=0.009) and mortality (from 10.4 to 5.7%, p=0.001). In modeling 25(OH)D as a log-transformed continuous variable, the greatest risk for hospitalization and death was observed at lower 25(OH)D concentrations. CONCLUSIONS: Continuous blood 25(OH)D concentrations are independently associated with COVID-19-related hospitalization and mortality in an inverse dose-response relationship in this large racially and ethnically diverse cohort of VA patients. Randomized controlled trials are needed to evaluate the impact of vitamin D supplementation on COVID-19-related outcomes.

Vitamin D: Newer Concepts of Its Metabolism and Function at the Basic and Clinical Level.

The interest in vitamin D continues unabated with thousands of publications contributing to a vast and growing literature each year. It is widely recognized that the vitamin D receptor (VDR) and the enzymes that metabolize vitamin D are found in many cells, not just those involved with calcium and phosphate homeostasis. In this mini review I have focused primarily on recent studies that provide new insights into vitamin D metabolism, mechanisms of action, and clinical applications. In particular, I examine how mutations in vitamin D metabolizing enzymes-and new information on their regulation-links vitamin D metabolism into areas such as metabolism and diseases outside that of the musculoskeletal system. New information regarding the mechanisms governing the function of the VDR elucidates how this molecule can be so multifunctional in a cell-specific fashion. Clinically, the difficulty in determining vitamin D sufficiency for all groups is addressed, including a discussion of whether the standard measure of vitamin D sufficiency, total 25OHD (25 hydroxyvitamin) levels, may not be the best measure-at least by itself. Finally, several recent large clinical trials exploring the role of vitamin D supplementation in nonskeletal diseases are briefly reviewed, with an eye toward what questions they answered and what new questions they raised.

Skeletal and Extraskeletal Actions of Vitamin D: Current Evidence and Outstanding Questions.

The etiology of endemic rickets was discovered a century ago. Vitamin D is the precursor of 25-hydroxyvitamin D and other metabolites, including 1,25(OH)2D, the ligand for the vitamin D receptor (VDR). The effects of the vitamin D endocrine system on bone and its growth plate are primarily indirect and mediated by its effect on intestinal calcium transport and serum calcium and phosphate homeostasis. Rickets and osteomalacia can be prevented by daily supplements of 400 IU of vitamin D. Vitamin D deficiency (serum 25-hydroxyvitamin D <50 nmol/L) accelerates bone turnover, bone loss, and osteoporotic fractures. These risks can be reduced by 800 IU of vitamin D together with an appropriate calcium intake, given to institutionalized or vitamin D-deficient elderly subjects. VDR and vitamin D metabolic enzymes are widely expressed. Numerous genetic, molecular, cellular, and animal studies strongly suggest that vitamin D signaling has many extraskeletal effects. These include regulation of cell proliferation, immune and muscle function, skin differentiation, and reproduction, as well as vascular and metabolic properties. From observational studies in human subjects, poor vitamin D status is associated with nearly all diseases predicted by these extraskeletal actions. Results of randomized controlled trials and Mendelian randomization studies are supportive of vitamin D supplementation in reducing the incidence of some diseases, but, globally, conclusions are mixed. These findings point to a need for continued ongoing and future basic and clinical studies to better define whether vitamin D status can be optimized to improve many aspects of human health. Vitamin D deficiency enhances the risk of osteoporotic fractures and is associated with many diseases. We review what is established and what is plausible regarding the health effects of vitamin D.

Vitamin D Assays.

The number of requests for vitamin D metabolite measurements has increased dramatically over the past decade leading commercial laboratories to develop rapid high throughput assays. The measurement of 25-hydroxyvitamin D (25[OH]D) and to a lesser extent 1,25-dihydroxyvitamin D (1,25[OH]2D) dominates these requests, but requests for multiple metabolite measurements in the same sample are also increasing. The most commonly used methods include immunoassays and liquid chromatography/mass spectrometry (LC-MS). Each method has its advantages and disadvantages, but with improvements in technology, especially in LC-MS, this method is gaining ascendance due to its greater precision and flexibility. The use of standards from the National Institutes of Standards and Technology has substantially reduced the variability from laboratory to laboratory, thereby improving the reliability of these measurements. Although the current demand is for measurement of total vitamin D metabolite levels, these metabolites circulate in blood tightly bound to vitamin D binding protein (DBP) and albumin with less than 1% free. The free concentration may be a more accurate indicator of vitamin D status especially in individuals with DBP levels that deviate from the normal population. Thus, methods to measure the free concentration at least of 25(OH)D are becoming available and may supplement if not replace measurements of total levels.

Vitamin D Prevents Sunburn: Tips for the Summer?

In the article by Scott et al, a high dose of vitamin D attenuated the inflammatory response to UV radiation in a small group of normal volunteers. The best results were in those subjects who had the greatest increase in circulating 25hydroxyvitamin D. Using microarray analyses these subjects showed a reduction in the expression of inflammatory markers with an increase in markers of skin barrier repair.

p120-Catenin Is Required for Dietary Calcium Suppression of Oral Carcinogenesis in Mice.

Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-ß-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low, or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation, and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. J. Cell. Physiol. 232: 1360-1367, 2017. © 2016 Wiley Periodicals, Inc.

Response of Vitamin D Concentration to Vitamin D3 Administration in Older Adults without Sun Exposure: A Randomized Double-Blind Trial.

OBJECTIVES: To determine the dose-response relationship between 25-hydroxyvitamin D (25(OH)D) and supplemental vitamin D3 in elderly nursing home residents. DESIGN: Randomized double-blind investigation. SETTING: Nursing home. PARTICIPANTS: Of 81 women (n=51) and men (n=30) (mean age 87.4±8) enrolled, 72 completed the study. INTERVENTION: Sixteen weeks of oral vitamin D3 at 800, 2,000, or 4,000 IU/d or 50,000 IU/wk. MEASUREMENTS: The main outcome was 25(OH)D concentrations (tandem mass spectrometry) after 16 weeks. Free 25(OH)D and intact parathyroid hormone (iPTH) were also analyzed. Safety monitoring of calcium and estimated glomerular filtration rate was performed, and adherence and clinical status were measured. RESULTS: 25(OH)D concentrations increased with dose (P<.001) and were higher with 50,000 IU/wk (P<.001) than other doses and with 4,000 IU/d than 800 or 2,000 IU/d, but 800 IU and 2,000 IU/d did not differ. One subject receiving 800 IU/d had concentrations less than 20 ng/mL. All subjects receiving more than 2000 IU/d had concentrations of 20 ng/mL and greater. Free 25(OH)D concentrations rose with total 25(OH) vitamin D. Total and free 25(OH)D were related to calcium concentrations; only free 25(OH)D was related to iPTH. CONCLUSION: 25(OH)D increased linearly with 800 to 4,000 IU/d and 50,000 IU/wk of vitamin D3, without a ceiling effect. Data suggest that some elderly adults will require more than 800 IU/d of vitamin D3 to ensure adequate vitamin D levels. Changes in 25(OH)D with vitamin D3 were related to starting concentrations (greatest with the lowest concentrations and unchanged with 800 and 2,000 IU/d if 20-40 ng/mL). Relationships between serum calcium and iPTH and free 25(OH)D suggest the potential for free 25(OH)D in defining optimal 25(OH)D concentrations.

Total 25(OH) vitamin D, free 25(OH) vitamin D and markers of bone turnover in cirrhotics with and without synthetic dysfunction.

BACKGROUND & AIMS: Current clinical assays for total 25-hydroxy (OH) vitamin D measure vitamin D bound to vitamin D-binding protein (DBP) and albumin plus unbound ('free') D. We investigated the relationship between total and free 25(OH)D with bone metabolism markers in normal (>3.5 g/dl) vs. low (≤3.5 g/dl) albumin cirrhotics. METHODS: Eighty-two cirrhotics underwent measurement of free and total 25(OH)D by immunoassay, DBP and markers of bone metabolism [intact parathyroid hormone (iPTH), C-telopeptide (CTX), bone-specific alkaline phosphatase (BSAP), osteocalcin, amino-terminal pro-peptide of type 1-collagen (P1NP)]. Pearson's coefficients assessed relevant associations. RESULTS: Cirrhotics with low (n = 54) vs. normal (n = 28) albumin had lower total 25(OH)D (12.1 vs. 21.7 ng/ml), free 25(OH)D (6.2vs.8.6 pg/ml) and DBP(91.4 vs. 140.3 µg/ml) [P < 0.01 for each]. iPTH was similar in low and normal albumin groups (33 vs. 28 pg/ml; P = 0.38), although serum CTX(0.46vs.0.28 ng/ml) and BSAP(31.7 vs. 24.8 µg/L) were increased (P < 0.01). An inverse relationship was observed between total 25(OH)D and iPTH in normal (r = -0.47, P = 0.01) but not low albumin cirrhotics (r = 0.07, P = 0.62). Similar associations were seen between free 25(OH)D and iPTH(Normal: r = -0.46, P = 0.01; Low: r = -0.03, P = 0.84). BSAP, osteocalcin and P1NP were elevated above the normal range in all cirrhotics but not consistently associated with total or free 25(OH)D. CONCLUSIONS: Cirrhotics with low vs. normal albumin have lower levels of DBP, total and free 25(OH)D. The expected relationship between total or free 25(OH)D with iPTH was observed in normal but not in low albumin cirrhotics, demonstrating that total 25(OH)D is not an accurate marker of bioactive vitamin D status in cirrhotics with synthetic dysfunction. Additional investigation into the role of vitamin D supplementation and its impact on bone mineral homoeostasis in this population is needed.

Vitamin D and bone.

All cells comprising the skeleton-chondrocytes, osteoblasts, and osteoclasts-contain both the vitamin D receptor and the enzyme CYP27B1 required for producing the active metabolite of vitamin D, 1,25 dihydroxyvitamin D. Direct effects of 25 hydroxyvitamin D and 1,25 dihydroxyvitamin D on these bone cells have been demonstrated. However, the major skeletal manifestations of vitamin D deficiency or mutations in the vitamin D receptor and CYP27B1, namely rickets and osteomalacia, can be corrected by increasing the intestinal absorption of calcium and phosphate, indicating the importance of indirect effects. On the other hand, these dietary manipulations do not reverse defects in osteoblast or osteoclast function that lead to osteopenic bone. This review discusses the relative importance of the direct versus indirect actions of vitamin D on bone, and provides guidelines for the clinical use of vitamin D to prevent/treat bone loss and fractures.

The nonskeletal effects of vitamin D: an Endocrine Society scientific statement.

Significant controversy has emerged over the last decade concerning the effects of vitamin D on skeletal and nonskeletal tissues. The demonstration that the vitamin D receptor is expressed in virtually all cells of the body and the growing body of observational data supporting a relationship of serum 25-hydroxyvitamin D to chronic metabolic, cardiovascular, and neoplastic diseases have led to widespread utilization of vitamin D supplementation for the prevention and treatment of numerous disorders. In this paper, we review both the basic and clinical aspects of vitamin D in relation to nonskeletal organ systems. We begin by focusing on the molecular aspects of vitamin D, primarily by examining the structure and function of the vitamin D receptor. This is followed by a systematic review according to tissue type of the inherent biological plausibility, the strength of the observational data, and the levels of evidence that support or refute an association between vitamin D levels or supplementation and maternal/child health as well as various disease states. Although observational studies support a strong case for an association between vitamin D and musculoskeletal, cardiovascular, neoplastic, and metabolic disorders, there remains a paucity of large-scale and long-term randomized clinical trials. Thus, at this time, more studies are needed to definitively conclude that vitamin D can offer preventive and therapeutic benefits across a wide range of physiological states and chronic nonskeletal disorders.

The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A.

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of ß-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Vitamin D: newly discovered actions require reconsideration of physiologic requirements.

Vitamin D is not just for preventing rickets and osteomalacia. Recent findings in animal experiments, epidemiologic studies and clinical trials indicate that adequate vitamin D levels are important for cancer prevention, controlling hormone levels and regulating the immune response. Although 25 hydroxyvitamin D (25OHD) levels >10 ng/ml can prevent rickets and osteomalacia, these levels are not sufficient to provide these more recently discovered clinical benefits. Rather, levels of 25OHD >30 ng/ml are generally recommended. Determining optimal levels of 25OHD and the amount of vitamin D supplementation required to achieve those levels for the numerous actions of vitamin D will only be established with additional trials. In this review, these newer applications are summarized and therapeutic considerations are provided.

Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes.

Extracellular Ca(2+) (Ca(2+)(o)) is a critical regulator that promotes differentiation in epidermal keratinocytes. The calcium sensing receptor (CaR) is essential for mediating Ca(2+) signaling during Ca(2+)(o)-induced differentiation. Inactivation of the endogenous CaR-encoding gene CASR by adenoviral expression of a CaR antisense cDNA inhibited the Ca(2+)(o)-induced increase in intracellular free calcium (Ca(2+)(i)) and expression of terminal differentiation genes, while promoting apoptosis. Ca(2+)(o) also instigates E-cadherin-mediated cell-cell adhesion, which plays a critical role in orchestrating cellular signals mediating cell survival and differentiation. Raising Ca(2+)(o) concentration ([Ca(2+)](o)) from 0.03 to 2 mm rapidly induced the co-localization of alpha-, beta-, and p120-catenin with E-cadherin in the intercellular adherens junctions (AJs). To assess whether CaR is required for the Ca(2+)(o)-induced activation of E-cadherin signaling, we examined the impact of CaR inactivation on AJ formation. Decreased CaR expression suppressed the Ca(2+)(o)-induced AJ formation, membrane translocation, and the complex formation of E-cadherin, catenins, and the phosphatidylinositol 3-kinase (PI3K), although the expression of these proteins was not affected. The assembly of the E-cadherin-catenin-PI3K complex was sensitive to the pharmacologic inhibition of Src family tyrosine kinases but was not affected by inhibition of Ca(2+)(o)-induced rise in Ca(2+)(i). Inhibition of CaR expression blocked the Ca(2+)(o)-induced tyrosine phosphorylation of beta-, gamma-, and p120-catenin, PI3K, and the tyrosine kinase Fyn and the association of Fyn with E-cadherin and PI3K. Our results indicate that the CaR regulates cell survival and Ca(2+)(o)-induced differentiation in keratinocytes at least in part by activating the E-cadherin/PI3K pathway through a Src family tyrosine kinase-mediated signaling.

Vitamin D insufficiency/deficiency in gastrointestinal disorders.

Vitamin D and calcium are critical for skeletal health. Their absorption from the intestine is negatively impacted by a number of gastrointestinal diseases and surgical procedures, leading to osteoporosis and/or osteomalacia. Diseases of the liver can impact the metabolism of vitamin D to its circulating form, 25(OH)D, as well as the production of carrier proteins, albumin and vitamin D-binding protein, that may alter the delivery of 25(OH)D and its active metabolite 1,25(OH)(2)D to target tissues, including the skeleton, again leading to bone disease. The clinician evaluating a patient with apparent osteoporosis and vitamin D deficiency/ insufficiency needs to consider a gastrointestinal etiology. Similarly, the clinician evaluating a patient with a gastrointestinal disorder needs to evaluate that patient for vitamin D deficiency and bone disease. Treatment involves adequate vitamin D and calcium supplementation to achieve normal serum 25(OH)D, PTH, and serum and urine calcium levels.

Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes.

Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.