Vitamin D-responsive SGPP2 variants associated with lung cell expression and lung function

Vitamin D is of interest in relation to a number of health outcomes, with putative function beyond its classical role in maintaining bone health. The active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)₂D], when bound to the vitamin D receptor (VDR), regulates the expression of genes in many molecular pathways, including inflammation, cell proliferation, cell death, and tissue-remodeling pathways [1]. Serum 25-hydroxyvitamin D [25(OH)D] is the primary circulating biomarker of vitamin D status, and recent national survey data in the U.S. indicate 32% of Americans are at risk of vitamin D inadequacy or deficiency, defined as 30–49 nmol/L and <30 nmol/L serum 25(OH)D, respectively [2, 3].

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States, and is a large and growing burden on health care [4]. While smoking is the primary risk factor for rapid lung function decline and development of COPD, about 15% of individuals who have never smoked develop COPD and not all smokers succumb, implicating other factors, such as genetic, dietary, and lifestyle factors, in lifetime lung function patterns and disease risk [5].

Recent evidence indicates that vitamin D, as a steroid hormone capable of influencing gene expression, may be a determinant of lung function [6]. A cross-sectional study in the National Health and Nutrition Examination Survey (NHANES) III reported a strong positive association between serum 25(OH)D and lung function, with clinically relevant effect sizes for forced expiratory volume in the first second (FEV₁) and forced vital capacity (FVC) [7]. However, a subsequent cross-sectional study in the U.K. reported no association between serum 25(OH)D and FEV₁[8]. Causal inferences are limited in the cross-sectional design, effect estimates may be biased by uncontrolled confounders such as physical activity, and, furthermore, comparisons are limited by differences in the range in serum 25(OH)D between studies. Investigations of serum 25(OH)D or high-dose vitamin D supplementation in relation to the risk of exacerbations in COPD patients reported overall null findings [9, 10]. However, vitamin D supplementation led to a statistically significant reduction in COPD exacerbations in the subgroup with severe vitamin D deficiency at the study baseline (serum 25(OH)D < 10 ng/mL) [9], underscoring the importance of considering the potential to benefit in studies of nutritional supplementation.

In vitro animal and cell culture studies demonstrate that vitamin D-responsive genes play a role in airway remodeling and inflammation, which are key processes in the pathogenesis of COPD [11, 12]. However, few studies directly investigate mechanisms for vitamin D’s effect in vivo, which would strengthen the causal inference of population-level association studies. Furthermore, most experimental work to date has focused on effects of the active metabolite of vitamin D, 1,25-dihydroxyvitamin D. This metabolite is generated in the kidney for systemic circulation, and in many tissues, including lung [13]. It is not yet established whether the population-level range in serum 25-hydroxyvitamin D, the primary biomarker for vitamin D status in humans, is associated with effects similar to those seen in vitro for 1,25-hydroxyvitamin D.

We used an interdisciplinary approach to investigate the mechanisms through which vitamin D affects lung function. Genes with in vitro evidence of vitamin D regulation were studied to assess whether serum 25(OH)D concentration was associated with gene expression in lung epithelial tissues sampled from free-living humans. Identified genes were investigated in a study of expression quantitative trait loci (eQTL) in human lung epithelial cells to assess if genetic variation affects gene expression. Also, identified genes were investigated in an epidemiologic cohort study in relation to pulmonary function phenotypes. We hypothesized that serum 25(OH)D affects expression of vitamin D-responsive genes by modulating levels of active 1,25(OH)₂D in lung tissue, and that variants in candidate genes directly regulated by 1,25(OH)₂D in lung tissue are associated with FEV₁ and FEV₁/FVC, the key parameters used for COPD diagnosis and staging.

Using an interdisciplinary genomics approach we investigated vitamin D and lung outcomes. SGPP2, a phosphatase involved in the sphingosine-1-phosphate signaling pathway, was identified in all stages of the study as a promising candidate gene contributing to vitamin D-mediated associations with lung function. SGPP2 is differentially expressed in vivo in lung epithelial cells by serum 25(OH)D. eQTL analysis demonstrates that sequence variants in SGPP2 are associated with lung cell gene expression. Although the eQTL finding does not prove that vitamin D regulation affects gene expression, the location of associated variants in regulatory regions supports the hypothesis of vitamin D regulation. Furthermore, a group of 3 linked SNPs in the SGPP2 promoter region are associated with lower FEV₁, a reduced FEV₁/FVC ratio, and a 2–3 fold increased risk of airflow obstruction in African-Americans, suggesting that a causal variant in this region may affect SGPP2 function and/or vitamin D binding, and, consequently, lung outcomes. Additionally, a SNP in SGPP2 is associated with FEV₁ in Health ABC European-Americans and SGPP2 variants were also associated with FEV₁ in the Framingham Heart Study, confirming effects across racial groups and in two cohort studies. This multi-faceted approach identifies putative mechanistic pathways for observed vitamin D—lung function associations while reducing the chance of false positive results.

SGPP2 plays a key role in the sphingolipid signaling pathway through dephosphorylation of sphingosine-1-phosphate (S1P) to sphingosine, which is then converted to ceramide or back to sphingosine-1-phosphate by other enzymes [25]. Sphingosine-1-phosphate acts as both an intracellular and extracellular signaling molecule, and regulates critical cell processes including apoptosis, cell growth, and immune function [25, 26]. Altered sphingolipid concentrations have important ramifications for lung function; ceramide concentrations are elevated in COPD, contributing to lung alveolar destruction [25]. Little research exists on SGPP2, although a 2006 paper showed that SGPP2 is up-regulated in response to inflammatory stimuli in endothelial cells, suggesting a possible role in mediating inflammation in lung tissue [27]. However, SGPP2’s biological function to alter sphingosine-1-phosphate concentrations suggests that this gene contributes to the regulation of sphingolipid signaling pathways in lung tissue.

We identified several additional genes, namely DAPK1, KCNS3, and FSTL1, and all three had mechanistic links to lung function identified through gene ontology analysis and literature reviews (Additional files 11 and 12). Expression of all three genes was strongly associated with serum 25(OH)D, and variants in these genes were associated with pulmonary function in the Health ABC cohort study. However, variants were not replicated in the Framingham Heart Study, nor were there observed eQTL associations. DAPK1, which is down-regulated by 1,25(OH)₂D both in vivo and in vitro, is a pro-apoptotic kinase linked to cytoskeletal remodeling and regulation of inflammatory gene expression in macrophages [28, 29]. SNPs in KCNS3, which encodes a voltage-gated potassium channel protein, were associated with airway hyperresponsiveness in past studies [30], which is of interest given postulated associations of airways hyperresponsiveness with an accelerated rate of FEV₁ decline and risk of COPD [31]. FSTL1 up-regulates pro-inflammatory cytokines; in mice, the highest expression level is in lung [32]. Dexamethasone, which is a glucocorticoid used to treat both asthma and COPD, is associated with expression of both KCNS3 and FSTL1; interestingly, there are striking similarities in the effects of dexamethasone and 1,25-dihydroxyvitamin on the expression of these genes. The combination of 1,25-dihydroxyvitamin D with dexamethasone was investigated in vitro as an anti-inflammatory treatment; our results suggest the strong possibility of synergistic effects for this treatment combination (Additional file 12 for references).

A major strength of this study is that it translates in vitro animal and cell culture studies to an in vivo study, and then extends to study population-level SNP associations with lung phenotypes, which are partially replicated in an independent cohort. The multi-stage approach identified SGPP2 as a promising vitamin D-responsive gene for further study. The demonstration of differential gene expression in lung tissue associated with the physiologic range of 25-hydroxyvitamin D in a diverse sample of free-living humans confirms in vitro studies, and, while our study does not manipulate vitamin D, the in vivo evidence of association is novel. The Health ABC population-based cohort study included high-quality spirometry, detailed information on confounding factors such as smoking and population stratification, and comprised 40% African-American participants, thus allowing consideration of this understudied population in genomic research. FEV₁ is a predictor of all-cause mortality [33], and thus SNP—FEV₁ associations are clinically relevant. Although associations between SNPs and the FEV₁/FVC ratio were also investigated, the associations were not as strong as for FEV₁. Thus, vitamin D may have a stronger association with overall lung health versus the risk of COPD. This study identifies plausible biological mechanisms that support a true effect of vitamin D on lung function, and will help to guide the design and analysis of randomized controlled intervention trials of the role of vitamin D in lung disease.

Given that the microarray analysis was used exclusively as a candidate screen, limitations including the lack of qPCR confirmation (not possible due to sample volume limitations), use of nominal P values, and the lack of race-stratified analysis (not possible due to sample size limitations) are less of a concern. As expected, the proportion of participants in the race/ethnicity groups varied by tertile of serum 25(OH)D given the role of skin pigmentation in vitamin D synthesis in response to sunlight [2]. Race may either confound the serum 25(OH)D—gene expression association, or, race may be a causal antecedent variable that, in part, causes serum 25(OH)D concentration and, in turn, differences in gene expression; adjusting for race may be an over-adjustment. Of note, in regressions adjusted for race the regression coefficients for the serum 25(OH)D—gene expression association were similar to unadjusted analyses.

While the studies were all cross-sectional, which limits causal inference, the harmony of findings across different designs partly mitigates this concern. Although it would have been ideal to use the same samples in all studies (that is, expression, eQTL and SNP—lung function studies), practical limitations led to the use of different samples in each phase. Finally, although gene-level replication was observed for SGPP2 and DAPK1, the specific SNPs associated with FEV₁ in Health ABC did not reach statistical significance in FHS. We hypothesize that the SGPP2 SNPs identified in the two cohort studies may be tagging the same unknown causal variant(s) or there may be multiple SGPP2 regulatory regions associated with lung function. Additionally, the strongest SNP—lung function associations in Health ABC were in African-Americans, and, because FHS includes only European Americans, the replication was partial. In summary, SNPs in SGPP2 were statistically significantly associated with lung outcomes after FDR multiple testing adjustment and a highly statistically significant lung eQTL was identified for SGPP2; SGPP2 emerged as a clear candidate in all stages of this work.

This study establishes for the first time that physiological concentrations of serum 25(OH)D are associated with differences in gene expression in human lung tissue, and that candidate vitamin D responsive genes are associated with pulmonary function outcomes. We hypothesize that genetic variants associated with pulmonary function in our study affect binding of the VDR/RXR heterodimer to the genome; however, further studies are needed to map lung tissue-specific regulatory regions. Recent evidence shows that vitamin D regulatory elements (VDREs) are located both proximal and distal to vitamin D-responsive genes at promoter regions and enhancer regions, respectively, and that VDR/RXR binding is cell-type specific [34]. This emphasizes the importance of genome-wide VDR/RXR mapping in lung cells to identify regulatory regions [34]. Additionally, in vitro studies of bronchial epithelial cells to directly assess gene expression changes due to vitamin D would contribute to the current understanding. Overall, the results of our study identify putative mechanisms through which vitamin D may affect lung function and, suggest a physiological range for 25-hydroxyvitamin D at which differential responses occur at the molecular level. Demonstrated associations strengthen the evidence for monitoring serum 25(OH)D concentrations in individuals at risk of rapid decline in lung function.