Vitamin D Modulates Prostaglandin E2 Synthesis and ... - ATS Journals

ORIGINAL RESEARCH Vitamin D Modulates Prostaglandin E2 Synthesis and Degradation in Human Lung Fibroblasts Xiangde Liu1, Amy Nelson1, Xingqi Wang1, Maha Farid1, Yoko Gunji1, Jun Ikari1, Shun Iwasawa1, Hesham Basma1, Carol Feghali-Bostwick2, and Stephen I. Rennard1 1

Pulmonary, Critical Care, Sleep and Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska; and 2Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Abstract Vitamin D insufficiency has been increasingly recognized in the general population worldwide and has been associated with several lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections. Fibroblasts play a critical role in tissue repair and remodeling, which is a key feature of COPD and asthma. Fibroblasts modulate tissue repair by producing and modifying extracellular matrix components and by releasing mediators that act as autocrine or paracrine modulators of tissue remodeling. The current study was designed to investigate if vitamin D alters fibroblast release of key autocrine/paracrine repair factors. First, we demonstrated that human fetal lung (HFL)-1 cells express the vitamin D receptor (VDR) and that vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] induce VDR nuclear translocation and increase VDR-DNA binding activity. We next demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced prostaglandin (PG)E2 production by human lung fibroblasts (HFL-1) but had no effect on transforming growth factor b1, vascular endothelial growth factor, or fibronectin production. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly

Vitamin D has long been known for its effects on calcium homeostasis and skeletal health. Recently, its noncalcium/nonskeletal effects have received increasing attention. In this regard, it has been reported that vitamin D deficiency is linked to decline of lung function, impaired immunity, and enhanced inflammation (1–5).

inhibited IL-1b–induced microsomal PGE synthase (mPGES)-1 expression; in contrast, all three forms of vitamin D stimulated 15-hydroxy PG dehydrogenase, an enzyme that degrades PGE2. Cyclooxygenase-1 and -2 and the other two PGE2 synthases (mPGES-2 and cytosolic PGE synthase) were not altered by vitamin D, 25(OH)D, or 1,25(OH)2D. Finally, the effect of PGE2 inhibition by 25(OH)D was observed in adult lung fibroblasts. These findings suggest that vitamin D can regulate PGE2 synthesis and degradation and by this mechanism can modulate fibroblast-mediated tissue repair function. Keywords: fibroblasts; vitamin D; PGE2

Clinical Relevance Findings of this study suggest that vitamin D can regulate prostaglandin E2 synthesis and degradation, and that by this mechanism, vitamin D can modulate fibroblast-mediated tissue repair function in diseases such as chronic obstructive pulmonary disease and asthma.

Vitamin D can be obtained from food or by the action of sunlight on skin. Vitamin D is hydroxylated at position 25 in the liver by 25-hydroxylase into 25hydroxyvitamin D [25(OH)D] and is further hydroxylated in the kidney by 1ahydroxylase (CYP27B1) (6). Classically, 1,25-dihydroxyvitamin D [1,25(OH)2D] is

considered to be the active form of vitamin D that binds to the vitamin D receptor (VDR) and leads to nuclear translocation and modulation of gene transcription (6). Recent studies have indicated that 25(OH) D can bind to VDR and regulate gene expression (7, 8). A direct effect of vitamin D has not been reported.

( Received in original form May 9, 2013; accepted in final form July 31, 2013 ) This work was supported by the Larson Endowment from the University of Nebraska Medical Center. Correspondence and requests for reprints should be addressed to Xiangde Liu, M.D., Ph.D., University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, NE 68198-5910. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 50, Iss 1, pp 40–50, Jan 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0211OC on August 13, 2013 Internet address: www.atsjournals.org

40

American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 1 | January 2014

ORIGINAL RESEARCH Lung fibroblasts play an important role in airway tissue repair and remodeling. Insufficient tissue repair mediated by lung fibroblasts may contribute to the development of COPD and emphysema (9, 10). In contrast, excessive fibroblastmediated repair can lead to fibrosis of the pulmonary parenchyma or airways. Studies have suggested that overproduction of prostaglandin (PG)E2 by the fibroblasts from patients with COPD may be associated with the development of COPD (10–12), whereas a lack of PGE2 may be related to interstitial or airway fibrosis (13–16). Because low vitamin D levels have been reported in COPD, we entertained the hypothesis that vitamin D deficiency may lead to reduced fibroblast-mediated repair functions and might do so by modulating the release of factors known to augment or inhibit fibroblast-mediated repair. To assess this, we determined VDR expression in human lung fibroblasts and the effect of vitamin D on fibroblast release of transforming growth factor (TGF)-b1, fibronectin, vascular endothelial growth factor (VEGF), and PGE2, which are factors that augment repair mediated by fibroblasts or other cells (TGF-b1, fibronectin, and VEGF) or inhibit fibroblast repair functions (PGE2). In the current study, we found that human lung fibroblasts express VDR and that all three forms of vitamin D induced VDR nuclear translocation and DNA binding. All three forms of vitamin D reduced PGE2 levels in media of human lung fibroblast cultures. We also explored the mechanism by which form of vitamin D modulates PGE2 levels. Neither vitamin D, 25(OH)D, nor 1,25(OH)2D affected the expression of COX-1 or COX-2, but all three forms of vitamin D inhibited inducible microsomal PGE synthase (mPGES)-1 protein and mRNA expression. In contrast, mPGES-2 and cytosolic PGES were unaltered by all three forms of vitamin D. All three forms of vitamin D also stimulated protein and mRNA expression of 15-hydroxy PG dehydrogenase (15PGDH), an enzyme that degrades PGE2 into 15-keto-PGE2.

Materials and Methods Materials

Vitamin D3 (vitamin D, or cholecalciferol), PGE2, anti–b-actin antibody, and

proteinase inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO). 25(OH)D and 1,25(OH)2D were purchased from TOCRIS Bioscience (Ellisville, MO). TGF-b1 ELISA duo-sets, VEGF ELISA duo-sets, and IL-1b were purchased from R&D Systems (Minneapolis, MN). Anti–COX-1 antibody, anti–COX-2 antibody, anti-VDR antibody, negative control small interfering RNA (siRNA), and siRNA-targeting VDR (VDRsiRNA) or 15-PGDH (PGDH-siRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The PGE2 enzyme immunoassay (EIA) kit, anti–mPGES-1, anti–mPGES-2, anti–cytosolic PGE synthase (cPGES), and anti–15-PGDH antibodies were purchased from Cayman Chemical (Ann Arbor, MI). Anti-TATA binding protein (nuclear protein loading control, ab818) and anti–a-smooth muscle actin antibody (ab7817) were purchased from Abcam, Inc. (Cambridge, MA). Cell Culture

Human fetal lung (HFL) fibroblasts, HFL-1, WI38, and IMR-90 were purchased from American Type Culture Collection (Rockville, MD). Normal adult lung fibroblasts were cultured from the lungs of unused donor lungs under an Institutional Review Board–approved protocol at University of Pittsburgh (17). All of the fibroblasts were trypsinized and passaged in routine cell culture using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 50 U/ml penicillin/streptomycin, and 50 mg/ml fungizone. HFL-1 cells were used from passages 15 through 20. Adult lung fibroblasts were used from passages 4 through 7. TGF-b1, Fibronectin, VEGF, and PGE2 Quantification

To assess the effect of vitamin D on fibroblast release of TGF-b1, fibronectin, VEGF, and PGE2, fibroblasts were plated in monolayer (2 3 105 cells/ml, 1 ml per well of a 12-well plate) in 10% FCS-DMEM and cultured for 2 days. Cells were then treated with 1 ml per well serum-free DMEM (SF-DMEM) 6 ethanol (EOH) (1:1,000 as control), vitamin D, 25(OH)D, or 1,25(OH)2D. After 2 days, media were harvested for ELISA (TGF-b1, VEGF, and fibronectin) or EIA (PGE2) as described below. Cells were trypsinized and counted with a Coulter Counter.

Liu, Nelson, Wang, et al.: Vitamin D Regulates PGE2 in Lung Fibroblasts

Total and active TGF-b1 were quantified following the manufacturer’s instructions with a brief modification as described previously (18). Fibronectin was quantified using a competitive ELISA as described previously (18). VEGF was quantified using an ELISA as described previously (19). PGE2 was quantified using an EIA kit following the manufacturer’s instructions (Cayman Chemical). Immunoblotting

Immunoblotting for COX-1, COX-2, mPGES-1, mPGES-2, cPGES, and 15PGDH was performed with whole cell lysates. Briefly, cells were plated into 60-mm dishes in 10% FCS-DMEM and cultured until nearly confluent. Cells were then treated with SF-DMEM supplemented with EOH (1:1,000 dilution as vehicle control), vitamin D, 25(OH)D, or 1,25(OH)2D in the presence or absence of IL-1b. After 24 hours in culture, cells were washed once with cold PBS and harvested with cell lysis buffer (35 mM Tris HCl [pH 7.4], 0.4 mM EGTA, 10 mM MgCl2, and 1:1,000 of proteinase inhibitor cocktail) or a commercially available kit for extracting nuclear proteins and cytoplasm (Active Motif, Carlsbad, CA). Cell lysates were briefly sonicated on ice and centrifuged at 10,000 3 g for 5 minutes at 48 C. SDSPAGE (10 or 12.5%) gels were prepared, and 5 to 10 mg per lane of cellular proteins were loaded. The resolved proteins were transferred to a polyvinylidene difluoride membrane and incubated with primary antibodies following the manufacturer’s instructions. After incubation with horseradish peroxidase–conjugated second antibodies, images were visualized and quantified with a Lumigen PS-3 (Beckman, Southfield, MI) and a Typhon Scanner (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). Real-Time RT-PCR

Fibroblasts were plated into 60-mm dishes and cultured until nearly confluent. Cells were then treated with SF-DMEM 6 EOH (1:1,000), vitamin D, 25(OH)D, or 1,25 (OH)2D in the presence or absence of IL-1b. After 24 hours of treatment, total RNA was extracted using Trizol (Invitrogen, Grand Island, NY) following the manufacturer’s instructions. RT was performed using a commercial kit following the manufacturer’s instructions (High Capacity cDNA Reverse Transcription Kit; 41

ORIGINAL RESEARCH Applied Biosystems, Invitrogen). Real-time PCR was conducted using presynthesized probe and primer sets purchased from Applied Biosystems (Invitrogen) following the manufacturer’s instructions. PCR with total volume of 25 ml for each reaction in duplicated assays for each sample was performed with a 7500 Real Time PCR Instrument (Invitrogen). Eukaryotic 18S rRNA Endogenous Control (Invitrogen) was used as endogenous control to quantify the expression of COX-1 (Hs00924808_m1), COX-2 (Hs00153133_m1), mPGES-1 (Hs01115610_m1), mPGES-2 (Hs00228159_m1), cPGES (Hs00832847_gH), and 15-PGDH (Hs00960586_g1), respectively. Data were normalized by the internal control and expressed as fold change versus EOH treatment. Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA) was performed using a kit from Panomics, Inc. (Redwood City, CA) following the manufacturer’s instructions. Briefly, cells were treated with the three forms of vitamin D for 24 hours, and nuclear proteins were extracted using a kit (Panomics, Inc.). Nuclear extract (5 mg) was incubated with 10 ng biotin-labeled VDR probe. Protein–DNA complexes were then resolved by nondenaturing PAGE. After transfer to Pall Biodyne B membrane (Pall Corp., East Hills, NY), proteins were immobilized with an ultraviolet crosslinker. After blocking, avidin-horseradish peroxidase was applied, and images were visualized by enhanced chemiluminescence (Amersham).

IL-1b (1 ng/ml). After 48 hours of treatment, media were harvested for PGE2 quantification by EIA, and the cell number was determined with a Coulter Counter. Statistical Analysis

All data were expressed as mean 6 SEM. Statistical comparisons of multigroup data were analyzed by ANOVA followed by Bonferroni’s (two-way) or Tukey’s (oneway) posttest correction using PRISM4 software.

Results Expression and Response of the VDR to Vitamin D, 25(OH)D, and 1,25(OH)2D

To determine the potential role for vitamin D in lung fibroblasts, we assessed VDR expression in HFL-1 cells, a primary culture of normal fetal lung fibroblasts. HFL-1 cells expressed VDR under control conditions (Figure 1A). All forms of vitamin D, in particular 25(OH)D, stimulated VDR expression by HFL-1 cells (Figure 1A). To determine the potential for the three forms of vitamin D to signal in HFL-1 cells, we assessed the ability of vitamin D, 25(OH)D, and 1,25(OH)2D to stimulate VDR nuclear translocation and to bind to DNA. All three forms of vitamin D significantly stimulated

nuclear translocation, with 25(OH)D demonstrating the most robust action (Figure 1B). Furthermore, vitamin D, 25 (OH)D, and 1,25(OH)2D stimulated VDRprotein binding activity, as evidenced by EMSA (Figure 1C). Although our study was not designed to determine the form of vitamin D that was active, the finding that all three forms of vitamin D were active suggested that fibroblasts may be able to metabolize vitamin D. To assess this, we examined expression of 25-hydroxylase (CYP2R1) and 1,25-dihydroxylase (CYP27B1) by HFL-1 cells by immunoblot. As expected, HFL-1 cells expressed 25-hydroxylase (CYP2R1) and 1,25-dihydroxylase (CYP27B1, data not shown). Modulation of HFL-1 Cell Release of Tissue Repair Mediators by Vitamin D, 25(OH)D, and 1,25(OH)2D

It is well known that PGE2 and TGF-b1, fibronectin, and VEGF play important roles in regulating fibroblast proliferation, differentiation, and tissue repair and remodeling. Therefore, the effect of vitamin D, 25(OH)D, and 1,25(OH)2D on PGE2, TGF-b1, fibronectin, and VEGF release by human lung fibroblasts (HFL-1 cells) was determined. Under control culture conditions, fibroblasts synthesize and release PGE2 into the culture media

RNA Interference

HFL-1 cells were plated in 100-mm dishes at a density of 2 3 106 cells per dish in 10% FCS-DMEM without penicillin/ streptomycin/fungizone. After 2 days, cells were transfected overnight with controlsiRNA, VDR-siRNA, or 15-PGDH-siRNA using lipofectamine-2000 and Opti-MEM medium (Invitrogen) following the manufacturer’s instructions. The final concentration of siRNA was 100 nM in 3 ml of Opti-MEM. Cells were then trypsinized and replated into 12-well plates at a density of 105/well/ml. After 2 days of culture in 10% FCS-DMEM, cells were treated with SF-DMEM supplemented with EOH (1:1,000 dilution as vehicle control) and 100 nM vitamin D, 25(OH)D, or 1,25(OH)2D in the presence or absence of 42

Figure 1. Vitamin D receptor (VDR) expression and its activation in human fetal lung (HFL)-1 cells. (A) VDR expression by HFL-1 cells. HFL-1 cells were cultured to nearly confluent and treated with 1 mM of vitamin D, 25-hydroxyvitamin D [25(OH)D], or 1,25-dihydroxyvitamin D [1,25(OH)2D] for 24 hours. Whole cell lysates were immunoblotted for VDR and b-actin. (B) Nuclear translocation of VDR. Nearly confluent HFL-1 cells were treated with 1 mM vitamin D, 25(OH)D, or 1,25(OH)2D for 24 hours. After washing with PBS, nuclear and cytoplasmic proteins were isolated using a commercial kit. Five micrograms of proteins were loaded, and immunoblotts for VDR, TATA binding protein (TBP, nuclear protein loading control), and b-actin were performed. (C) Electrophoretic mobility shift assay (EMSA) for VDR. Nuclear proteins were extracted using a commercial kit. EMSA was then performed following the manufacturer’s instruction. The arrow indicates VDR–DNA binding. Data presented are representative of three separate experiments.


ORIGINAL RESEARCH (174.3 6 9.0 pg/d/106 cells). Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited PGE2 release (70.4 6 21.4 pg/d/ 106 cells by vitamin D, 22.5 6 1.5 pg/d/106 cells by 25(OH)D, and 26.9 6 5.9 pg/d/106 cells by 1,25(OH)2D, respectively; P , 0.05 or P , 0.01) (Figure 2A). In contrast, vitamin D, 25(OH)D, and 25(OH)2D had no effect on fibronectin, TGF-b1, or VEGF release by HFL-1 cells (Figures 2B–2D). Similar results were observed in three-

dimensional collagen gel culture, in which all forms of vitamin D inhibited PGE2 release but had no effect on TGF-b1, fibronectin, or VEGF release (see Figure E1 in the online supplement). Finally, the effect of vitamin D, 25(OH)D, and 1,25(OH)2D on PGE2 release by lung fibroblasts was concentration dependent under both control conditions (Figure 2E) and after stimulation with IL-1b (Figure 2F).

To confirm that vitamin D inhibits PGE2 release through an action on the VDR, we used siRNA to suppress the VDR. Transfection of VDR-siRNA resulted in over 90% suppression of VDR without affecting cell survival and phenotype (data not shown). As expected, suppression of VDR by siRNA completely blocked the inhibitory effect of vitamin D, 25(OH)D, and 1,25(OH)2D on PGE2 release by HFL-1 cells (Figure 3). HFL-1 cells transfected with VDR-siRNA released more PGE2 compared with the cells transfected with control-siRNA (Figure 3), particularly in the presence of IL-b (P , 0.05) (Figure 3B). Next, we examined effect of vitamin D, 25(OH)D, and 1,25(OH)2D on fibroblastmediated collagen gel contraction and the role of PGE2 in mediating vitamin D modulation of fibroblast-mediated collagen gel contraction. Consistent with the inhibitory effect of vitamin D on PGE2 release, all three forms of vitamin D enhanced collagen gel contraction in a time-dependent (Figure E2A) and concentration-dependent manner (Figure E2B). Exogenous PGE2 partially but significantly blocked vitamin D augmentation of collagen gel contraction (Figure E2C), supporting the concept that PGE2 can inhibit contraction in the presence of vitamin D, 25(OH)D, and 1,25(OH)2D. Furthermore, after indomethacin pretreatment, 25(OH)D still induced augmented collagen gel contraction in a concentration-dependent manner, suggesting that vitamin D can augment contraction by mechanisms in addition to PGE2 inhibition (Figure E2D). Mechanistic Basis for Vitamin D, 25(OH)D, and 1,25(OH)2D Reduction of PGE2 Levels

Figure 2. Effect of vitamin D, 25(OH)D, and 1,25(OH)2D on prostaglandin (PG)E2, fibronectin, TGFb1, and vascular endothelial growth factor (VEGF) production by fibroblasts. HFL-1 cells were plated into 12-well plates at a density of 2 3 105 cells per well in 10% FCS-Dulbecco’s modified Eagle medium (DMEM). After 2 days of culture, cells were treated with 1 ml per well of serum-free DMEM (SF-DMEM) 6 ethanol (EOH, 1:1,000), 1 mM vitamin D, 1 mM 25(OH)D, or 1 mM 1,25(OH)2D. After 48 hours of treatment, media were harvested. PGE2 was quantified by EIA (A); fibronectin (B), TGF-b1 (C), and VEGF (D) were measured by ELISA. (E and F) HFL-1 cells were plated into 12-well plates as described above. Cells were treated with varying concentrations of vitamin D, 25(OH)D, or 1,25 (OH)2D in the absence (E) or presence (F) of IL-1b (1 ng/ml). After 48 hours of treatment, media were harvested and used for PGE2 quantification by EIA. Vertical axes: PGE2 amount (pg/d/106 cells). Horizontal axes: Concentrations of vitamin Ds (nM). Data presented are representative of four separate experiments. **P , 0.01 compared with EOH alone by one-ANOVA followed by Tukey’s test.


Several enzymes play important roles in PGE2 synthesis and metabolism. In this regard, cyclooxygenase (COX) enzymes and the three PGE synthases (PGES) can lead to PGE2 synthesis from arachidonic acid, whereas 15-PGDH is a key enzyme that degrades PGE2 by catalyzing oxidation of PGE2 into 15-keto-PGE2. Therefore, we examined the effect of vitamin D, 25(OH) D, and 1,25(OH)2D on COX-1 and COX-2 expression with or without IL-1b stimulation. As expected, the constitutively expressed COX-1 was easily detected under control conditions, and its expression was not affected by IL-1b. Also as expected, IL-1b stimulated COX-2 expression. 43

ORIGINAL RESEARCH

Figure 3. Role of VDR in mediating the inhibitory effect of vitamin D, 25(OH)D, and 1,25(OH)2D on PGE2 release. HFL-1 cells were transfected with control–small interfering RNA (siRNA) or VDR-siRNA. Cells were then treated with or without 100 nM vitamin D, 25(OH)D, or 1,25(OH)2D in the presence or absence of IL-1b (1 ng/ml) for 48 hours. PGE2 in the medium was quantified with EIA. (A) Without IL-1b stimulation. (B) With IL-1b stimulation. Vertical axes: PGE2 amount. Horizontal axes: Cells transfected with control-siRNA or VDR-siRNA. *P , 0.05. Data presented are representative of three separate experiments.

However, neither COX-1 nor COX-2 expression at the protein or mRNA level was altered by any of the tested forms of vitamin D [vitamin D, 25(OH)D and 1,25(OH)2D] with or without IL-1b stimulation (Figure 4).

We next examined the effect of vitamin D, 25(OH)D, and 1,25(OH)2D on the expression of the three forms of PGES: microsomal PGES (mPGES)-1, mPGES-2, and cPGES. Under control culture conditions, mPGES-1 mRNA (Figure 5A)

Figure 4. Effect of vitamin D, 25(OH)D, and 1,25(OH)2D on cyclooxygenase (COX)-1 and COX-2 expression. (A) mRNA expression of COX-1 and COX-2. HFL-1 cells were plated into 60-mm dishes in 10% FCS-DMEM. After 2 days culture, cells were treated with SF-DMEM 6 EOH (1:1,000), 1 mM vitamin D, 1 mM 25(OH)D, or 1 mM 1,25(OH)2D in the presence or absence of IL-1b (1 ng/ml). After 24 hours, total RNA was extracted using Trizol reagent. Real-time RT-PCR was conducted as described MATERIALS AND METHODS. (B) Immunoblot. HFL-1 cells were plated into 60-mm dishes and treated as described above. After 24 hours of treatment, whole cell lysate proteins were extracted and immunoblotted for COX-1, COX-2, and b-actin. (C) Densitometric analysis of COX-1 and COX-2 expression. The density of each blot was analyzed using Image J software. Vertical axis: Densitometry ratio versus IL-1b–treated sample. Horizontal axis: Treatment with various reagents. Hatched bars: COX-1. Solid bars: COX-2. Data presented are an average of three separate experiments.

44

and protein (Figure 5B) were barely detectable, whereas the mRNAs (Figure 5D) and proteins (Figure 5E) of mPGES-2 and cPGES were easily detected. As expected, protein expression of the inducible mPGES-1 was significantly stimulated by IL-1b (an average of 2.2 6 0.3-fold increase after stimulation with 1 ng/ml IL-1b; P , 0.05 compared with control) (Figures 5B and 5C), whereas expression of the constitutive mPGES-2 and cPGES was not significantly affected by IL-1b (Figures 5E and 5F). Protein expression for the inducible mPGES-1 was slightly inhibited by vitamin D, 25(OH)D, and 1,25(OH)2D when the cells were cultured under control conditions, although this did not reach statistical significance (Figures 5B and 5C). In the presence of IL-1b, which upregulates mPGES-1 expression, vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited mPGES-1 protein (P , 0.05 for all comparisons to IL-1b) (Figure 5C) and mRNA expression (P , 0.01 all comparisons to IL-1b) (Figure 5A). We next examined if vitamin D could modulate PGE2 degradation by regulating 15-PGDH. Using real-time RT-PCR, mRNA of 15-PGDH was easily detectible, and vitamin D, 25(OH)D, and 1,25(OH)2D significantly stimulated 15-PGDH mRNA expression (P , 0.05 or , 0.01) (Figure 6A). In contrast, IL-1b significantly inhibited 15-PGDH mRNA expression, which was partially but significantly blocked by 25(OH)D and 1,25(OH)2D (P , 0.05) (Figure 6A). Using immunoblot, we further demonstrated that protein of 15-PGDH was easily detectable under control culture conditions in HFL-1 cells (Figure 6B). Consistent with the effect on mRNA expression, vitamin D, 25(OH)D, and 1,25(OH)2D significantly stimulated 15-PGDH expression by HFL-1 cells (P , 0.05 compared with EOH control only) (Figures 6B and 6C). In contrast, IL-1b (1 ng/ml) significantly inhibited the expression of 15-PGDH by HFL-1 cells, which was partially but significantly blocked by vitamin D, 25(OH)D, or 1,25 (OH)2D (P , 0.05) (Figures 6B and 6C). To further confirm the role of 15PGDH in regulating PGE2 levels, HFL-1 cells were transfected with a negative control-siRNA or with 15-PGDH–siRNA followed by treatment with 100 nM vitamin D, 25(OH)D, or 1,25(OH)2D in the presence or absence of IL-1b (1 ng/ml).


ORIGINAL RESEARCH Transfection of control-siRNA or 15PGDH–siRNA with lipofecatmine did not affect cell growth. The 15-PGDH–specific siRNA significantly suppressed 15-PGDH expression (data not shown). In the absence of IL-1b, PGE2 levels in the media of cells lacking 15-PGDH was roughly 20% higher than that in cells transfected with controlsiRNA, but this was not statistically significant (Figure 7A). In contrast, the inhibitory effect of 25(OH)D and 1,25 (OH)2D on PGE2 was abolished in the cells transfected with 15-PGDH–siRNA (Figure 7A). When the cells were treated with IL-1b, the PGE2 level was significantly higher in the cells lacking 15-PGDH (P , 0.01) (Figure 7B). Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited PGE2 in the cells transfected with controlsiRNA or 15-PGDH–siRNA (P , 0.05) (Figure 7B). However, the magnitude of the inhibition by vitamin D and 1,25(OH)2D was less in the cells transfected with the 15-PGDH–siRNA compared with the cells transfected with con-siRNA (33.2 6 5.7% vs. 39.7 6 2.9% by vitamin D and 25.8 6 7.4% vs. 55.9 6 3.3% by 1,25(OH)2D, respectively) but was similar in magnitude inhibition by 25(OH)D (60.2 6 7.1 vs. 57.1 6 3.7%). Generalizability of Findings in HFL-1 Cells to Normal Adult Lung Fibroblasts

Figure 5. Effect of vitamin D, 25(OH)D, and 1,25(OH)2D on the expression of microsomal prostaglandin E synthase (mPGES)-1, mPGES-2, and cytosolic prostaglandin E synthase (cPGES). (A and D) mRNA expression. HFL-1 cells were plated into 60-mm dishes in 10% FCS-DMEM. After 2 days in culture, cells were treated with SF-DMEM 6 EOH (1:1,000), 1 mM vitamin D, 1 mM 25(OH)D, or 1 mM 1,25(OH)2D in the presence or absence of IL-1b (1 ng/ml). After 24 hours, total RNA was extracted using Trizol reagent. Real-time RT-PCR was conducted as described in MATERIALS AND METHODS. (B and E) Immunoblot. HFL-1 cells were plated into 60-mm dishes and treated as described above. After 24 hours of treatment, total cell lysate proteins were immunoblotted for mPGES-1, mPGES-2, cPGES, and b-actin as described in MATERIALS AND METHODS. (C and F) Densitometric analysis of mPGES-1, mPGES-2, and cPGES. Densitometric quantification was performed using Image J software. Vertical axes: Ratio versus EOH-treated sample. Horizontal axes: Treatment with various reagents. Data presented are an average of three separate experiments. *P , 0.05; **P , 0.01 by two-way ANOVA followed by Bonferroni correction.


To generalize the findings of vitamin D inhibition of PGE2 in lung fibroblast cultures, cells from fetal and adult lung were treated with 100 nM 25(OH)D for 48 hours. PGE2 levels in the medium were quantified by EIA and normalized by cell number. 25(OH)D inhibited PGE2 in the fibroblasts from fetal and adult lungs in the absence (161.8 6 39.1 vs. 72.4 6 12 pg/d/ 106 cells [n = 6]; P = 0.02) or presence of IL-1b (2,395 6 289 vs. 654.3 6 83.6 pg/d/ 106 cells [n = 6]; P , 0.01) (Figure 8).

Discussion In the current study, we have demonstrated that HFL-1 cells express VDR and that vitamin D, 25(OH)D, and 1,25(OH)2D stimulated nuclear translocation and DNAprotein binding activity of VDR. We also found that vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibit PGE2 in human lung fibroblast cultures in a concentration-dependent manner in 45

ORIGINAL RESEARCH

Figure 6. Effect of vitamin D, 25(OH)D, and 1,25(OH)2D on 15-hydroxy prostaglandin E2 dehydrogenase (15-PGDH) expression. (A) mRNA expression. HFL-1 cells were plated into 60-mm dishes in 10% FCS-DMEM. After 2 days in culture, cells were treated with SF-DMEM 6 EOH (1:1,000), 1 mM vitamin D, 1 mM 25(OH)D, or 1 mM 1,25(OH)2D in the presence or absence of IL-1b (1 ng/ml). After 24 hours, total RNA was extracted using Trizol reagent. Real-time RT-PCR was conducted as described in MATERIALS AND METHODS. (B) Immunoblot. HFL-1 cells were plated into 60-mm dishes and treated as described above. Total cell lysate proteins were immunoblotted for 15-PGDH and b-actin. (C) Densitometric analysis of 15-PGDH in monolayer and 3D culture. Vertical axes: Ratio versus EOH only. Horizontal axes: Treatment with various reagents. Data presented are an average of three separate experiments. *P , 0.05; **P , 0.01 by t test.

monolayer and three-dimensional collagen gel culture. Vitamin D, 25(OH)D, and 1,25 (OH)2D had no effect on TGF-b1, VEGF, and fibronectin production in monolayer or three-dimensional collagen gel culture. Suppression of VDR by siRNA abolished the inhibitory effect of vitamin D, 25(OH) D, and 1,25(OH)2D on PGE2 release by the lung fibroblasts, suggesting that VDR plays an important role in mediating vitamin D signaling in the lung fibroblasts. Although vitamin D, 25(OH)D, and 1,25(OH)2D significantly reduced levels of PGE2, there was no effect on COX-1, COX-2, mPGES-2, or cPGES expression. However, vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited mPGES-1, an inducible PGE2 synthase, and significantly stimulated 15PGDH, a key enzyme that degrades PGE2 into 15-keto–PGE2. Vitamin D3 (here simply called vitamin D) can be obtained through food or by synthesis in the epidermis in response to 46

ultraviolet B radiation. The first step of metabolic activation of vitamin D occurs through hydroxylation of 25-carbon by the enzyme called 25-hydroxylase, which occurs primarily in the liver, resulting in the formation of 25(OH)D. 25(OH)D, bound to vitamin D binding protein, is the major circulating form of vitamin D. Plasma 25(OH)D levels are commonly used as an indicator of vitamin D status. The second step of vitamin D metabolic activation is formation of 1,25dihydroxyvitamin D from 25(OH)D, which is catalyzed by 1a-hydroxylase primarily in the kidney (6). 1,25(OH)2D binds to the VDR, which forms a complex with the retinoid X receptor and transactivates vitamin D–responsive genes via vitamin D–responsive elements. In this manner, diverse physiologic processes, such as calcium homeostasis and cellular differentiation and proliferation, can be regulated (20). Recent studies have reported

that 1a-hydroxylase is also expressed in many cell types other than renal cells, including prostate, lung, colon, breast cells, and monocytes (21), and have suggested a role of 25(OH)D in these tissues, including prostate, mammary gland, and lungs. The 1,25(OH)2D produced by these extrarenal cells or tissues has been suggested to serve primarily as an autocrine/paracrine factor that can modulate cellular proliferation, differentiation and immune function (6). Vitamin D insufficiency [, 30 ng/ml 25(OH)D] has been increasingly recognized (22). Epidemiologic studies suggest that vitamin D deficiency [, 20 ng/ml 25(OH)D] might be associated with many lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), and respiratory tract infections (23). In this regard, studies have suggested that vitamin D deficiency is associated with poor lung function in susceptible populations, especially in asthma and COPD (3, 24, 25). The pathogenetic role of vitamin D deficiency in association with COPD is not fully understood. The current study, therefore, was designed to investigate if vitamin D plays a role in modulating fibroblast production of mediators that regulate lung tissue repair and remodeling. After airway injury, lung fibroblasts migrate to the wounded area and initiate tissue repair and remodeling. Many factors, including fibroblast products, regulate tissue repair functions. Among these are TGF-b1, PGE2, VEGF, and fibronectin. TGF-b1 and fibronectin are known to stimulate fibroblast-mediated repair, whereas PGE2 is an inhibitor of fibroblast tissue repair functions. VEGF mediates fibroblast regulation of endothelial cells. Thus, the effect of vitamin D on fibroblast release of TGF-b1, PGE2, VEGF, and fibronectin was investigated in the current study. Vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited PGE2 production by human lung fibroblasts, whereas production of TGF-b1, VEGF, and fibronectin was not affected by any of the vitamin Ds. This suggests vitamin D could shift the balance toward a more reparative milieu. Fibroblast-mediated collagen gel contraction is considered an in vitro model of tissue remodeling. Excessive “repair” may result in lung fibrosis, whereas insufficient repair may lead to emphysema (10). Here, we report that vitamin D, 25(OH)D, and 1,25(OH)2D augment


ORIGINAL RESEARCH

Figure 7. Suppression of 15-PGDH by siRNA and its effect on PGE2 level. HFL-1 cells were transfected with control-siRNA or 15-PGDH–siRNA as described in MATERIALS AND METHODS. Cells were treated in SF-DMEM 6 ethanol (1:1,000), 100 nM vitamin D, 25(OH)D, or 1,25(OH)2D in the presence or absence of 1 ng/ml IL-1b for 48 hours. Medium was harvested and PGE2 was quantified by EIA under control conditions (A) or in the presence of IL-1b (1 ng/ml) (B). Vertical axes: PGE2 level (pg/d/ 106 cells). Horizontal axes: Cells transfected with control-siRNA or 15-PGDH-siRNA. *P , 0.05; **P , 0.01. Data presented are representative of three separate experiments.

fibroblast-mediated collagen gel contraction. The ability of vitamin D to inhibit PGE2 production can account for

part of the augmented collagen gel contraction. However, augmented contraction was also observed in the

Figure 8. PGE2 release by fetal and adult lung fibroblasts and its inhibition by 25(OH)D. Three cell lines of fetal lung fibroblasts (HFL-1, WI38, and IMR90) and three adult lung fibroblasts isolated from normal donors were plated into 12-well tissue culture plate at a density of 2 3 105 cells per well in 10% FCS-DMEM. After 2 days in culture, cells were treated with 1 ml per well SF-DMEM 6 EOH (1:1,000) or 100 nM 25(OH)D in the presence or absence of IL-1b (1 ng/ml). After 48 hours of treatment, media were harvested. PGE2 was quantified by EIA. (A) PGE2 release under control conditions; (B) PGE2 release in the presence of IL-1b. Vertical axis: PGE2 (pg/d/106 cells). Horizontal axis: Cells were treated with ethanol or 25(OH)D. Data presented are representative of three separate experiments.


presence of indomethacin, which blocks PGE2 production. This suggests that vitamin D may augment collagen gel contraction by PGE2–independent mechanisms as well. Our results regarding collagen gel contraction differ from previous studies, although there are important methodological differences. Greiling and Thieroff-Ekerdt reported that 1,25(OH)2D inhibits skin fibroblast–induced collagen gel contraction (26), and Ramirez and colleagues reported that 1,25(OH)2D inhibits the profibrotic effect of TGF-b1, including inhibition of TGF-b1–augmented collagen gel contraction and a-smooth muscle actin expression (27). In the Greiling and Ramirez studies, however, fibroblasts were treated with 1,25(OH)2D in monolayer before casting into collagen gels. In our study, fibroblasts were cast into collagen gels followed by exposure to vitamin D, 25(OH)D, and 1,25(OH)2D during the contraction. We have found that these forms of vitamin D augmented collagen gel contraction (Figure E2) but had no effect on a-smooth muscle actin expression (Figure E3). Although the mechanisms for the differences between these studies remain to be further defined, there are many differences in fibroblast response in monolayer culture compared with three-dimensional gel culture. The key findings in the current study are that vitamin D inhibits PGE2 production by inhibiting mPGES-1 and augmenting 15PGDH expression. It is likely that the effects on collagen gel contraction reflect altered PGE2 production and other effects mediated by vitamin D. In vivo, 25(OH)D circulates at a concentration of 25 to 200 nM with a half-life of 15 days, whereas 1,25(OH)2D has a half-life of 15 hours (28). Consistent with these levels, we demonstrated that the inhibitory effects of vitamin D, 25 (OH)D, and 1,25(OH)2D on PGE2 were concentration dependent. Therefore, 10 to 100 nM of vitamin D, 25(OH)D, or 1,25 (OH)2D could significantly inhibit PGE2 release (29). PGE2 is produced and degraded by a series of enzymatic reactions. We therefore investigated role of enzymes that contribute to PGE2 metabolism. We found that neither of the cyclooxygenases (COX-1 and COX-2), which produce prostaglandin H (PGH) from arachidonic acid, were affected by vitamin D, 25(OH)D, or 47

ORIGINAL RESEARCH 1,25(OH)2D. This was true under control conditions and after IL-1b stimulation, which markedly induces COX-2. PGH is converted to PGE by one of three PGE synthases. Neither mPGES-2 nor cPGES, the constitutively expressed PGE2 synthase, was affected by any of the vitamin Ds. In contrast, the inducible microsomal PGE2 synthase (mPGES-1), which was barely detectable under control conditions, was significantly inhibited by vitamin D, 25 (OH)D, and 1,25(OH)2D when the cells were stimulated with IL-1b. We also examined the effect of vitamin D on the expression of the PGE2 degrading enzyme 15-PGDH. Under control conditions, vitamin D, 25(OH)D, and 1,25(OH)2D significantly stimulated 15PGDH expression. In contrast, IL-1b significantly inhibited 15-PGDH, and this inhibition was antagonized by vitamin D, 25(OH)D, and 1,25(OH)2D. These findings suggest that vitamin D, 25(OH)D, and 1,25(OH)2D also regulate PGE2 levels by stimulating 15-PGDH expression and increasing degradation of PGE2. Thus, vitamin D can modulate PGE2 by decreasing production through reduction of mPGES-1 and by increasing degradation via increasing 15-PGDH. The relative importance of the two mechanisms was not determined in the current study and likely varies with the local milieu. In the absence of IL-1b, suppression of 15-PGDH completely blocked the ability of vitamin D to reduce PGE2 levels. This is consistent with very low levels of mPGES-1 expression under our “control” conditions, in which the effect of vitamin D is likely mediated mostly by 15-PGDH. After stimulation with IL-1b, however, mPGES-1 is induced. In this milieu, suppression of 15-PGDH only partially blocked the effect of vitamin D in reducing PGE2 levels. This is consistent with the concept that mPGES1 plays a role in the presence of IL-1b and supports the concept that inhibition of PGE2 is multifactorial. In the current study, we assessed only lung fibroblasts. PGE2 is the major prostanoid produced by these cells. However, PGE2 is not the only potential product of PGH. In other cell types, PGD, PGF, prostacyclin, and thromboxane can be produced by the action of specific synthases. This raises the possibility that vitamin D, by reducing the activity of mPGES-1, could shift the balance from PGE2 to other prostanoids. This would be 48

further amplified by the induction of 15PGDH, which degrades PGE2. Because many physiologic functions are regulated in opposing ways by different prostanoids, this could result in a particularly potent effect of vitamin D. An effect of vitamin D on PGE2 metabolism has been reported in prostate cancer cells. Krishnan and colleagues (30) found that calcitriol [1,25(OH)2D] inhibited PGE2 synthesis and action by the following three mechanisms: (1) inhibition of COX-2 expression, (2) induction of 15PGDH, and (3) decrease of E-prostanoid receptors. In our study, although the induction of 15-PGDH by vitamin Ds was observed, inhibition of COX-2 expression, even in the presence of IL-1b, was not. However, we found that vitamin D, 25(OH) D, and 1,25(OH)2D significantly inhibited the inducible microsomal PGE synthase mPGES-1. These findings suggest that the mechanisms by which vitamin D regulates PGE2 may vary depending on cell type. PGE2 levels have been reported to be increased in the lower respiratory tract of patients with COPD. In this context, fibroblasts are a major source of PGE2 in the lung, and fibroblasts from patients with COPD overproduce PGE2 (10). This overproduction accounts, in part, for reduced repair responses in COPD fibroblasts. Vitamin D deficiency has been reported to be common among patients

with COPD. The importance of vitamin D depletion or deficiency in leading to overproduction of PGE2 is undetermined. However, the results of the current study suggest this as a potential mechanism. The current study also suggests that vitamin D repletion may be a strategy to reduce PGE2 levels in the lungs of patients with COPD. 1,25(OH)2D is the most active hormonal form of vitamin D; thus, 1,25 (OH)2D is the most extensively studied vitamin D metabolite. Recently, however, 25(OH)D has been reported to have direct effect in variety kinds of cells, including a chemopreventive effect in breast epithelial cells (7, 8), acceleration of fracture healing (31), and inhibition of prostate cancer cell proliferation (32). Furthermore, it has been reported that 25(OH)D is an agonistic VDR ligand (33) and could inhibit proliferation of prostatic stromal cells (34) and primary prostatic epithelial cells (32). Consistent with this report, we have found that not only 1,25(OH)2D but also the precursor forms of vitamin D and 25(OH)D inhibited PGE2 release by lung fibroblasts, although the effect of vitamin D is weaker than that of 25(OH)D and 1,25(OH)2D. Although the current study was not designed to investigate vitamin D metabolism by fibroblasts, lung fibroblasts express enzymes that can convert vitamin D to 25 (OH)D and 25(OH)D to 1,25-(OH)2D, providing a mechanism for precursors to have activity.

Figure 9. Schematic illustration of the PGE2 metabolism and its regulation by vitamin Ds. EP = Eprostanoid.


ORIGINAL RESEARCH In support of this concept, all three forms of vitamin D induced nuclear translocation of VDR and stimulated DNAprotein binding activity as evidenced by VDR-EMSA, suggesting that all three forms of vitamin D signal through VDR in HFL-1 cells. This was further supported by an RNA-interfering experiment demonstrating that suppression of VDR by siRNA completely abrogated the inhibitory effect of vitamin D, 25(OH)D, and 1,25(OH)2D on PGE2 release. However, VDR-siRNA per se resulted in up-regulation of PGE2 synthesis, suggesting that vitamin D may modulate “baseline” PGE2 production. The effect of vitamin D was particularly evident in the presence of IL-1b, which stimulates PGE2 production and induces COX-2 and mPGES-1. In the absence of IL-1b, siRNA

for VDR suppressed VDR expression. However, there is very little baseline expression of mPGES-1, and this is unchanged by the siRNA for VDR. 15PGDH is observed in the absence of IL-1b. However, a decrease was not observed in the presence of VDR-siRNA (Figure E4), suggesting that VDR modulation of the PGE2 biosynthetic pathway enzymes may require augmentation by IL-1b stimulation. This raises the possibility that the VDR signaling may interact specifically with IL1b signaling pathways. In summary, we have demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D reduce PGE2 levels in the medium of lung fibroblasts cultured in vitro through inhibiting mPGES-1 and stimulating 15-PGDH (Figure 9). Vitamin D inhibited

References 1. Janssens W, Lehouck A, Carremans C, Bouillon R, Mathieu C, Decramer M. Vitamin D beyond bones in chronic obstructive pulmonary disease: time to act. Am J Respir Crit Care Med 2009;179:630–636. 2. Sutherland ER, Goleva E, Jackson LP, Stevens AD, Leung DY. Vitamin D levels, lung function, and steroid response in adult asthma. Am J Respir Crit Care Med 2010;181:699–704. 3. Chishimba L, Thickett DR, Stockley RA, Wood AM. The vitamin D axis in the lung: a key role for vitamin d-binding protein. Thorax 2010;65: 456–462. 4. Liu N, Nguyen L, Chun RF, Lagishetty V, Ren S, Wu S, Hollis B, DeLuca HF, Adams JS, Hewison M. Altered endocrine and autocrine metabolism of vitamin D in a mouse model of gastrointestinal inflammation. Endocrinology 2008;149:4799–4808. 5. McMahon L, Schwartz K, Yilmaz O, Brown E, Ryan LK, Diamond G. Vitamin D-mediated induction of innate immunity in gingival epithelial cells. Infect Immun 2011;79:2250–2256. 6. Ebert R, Schutze N, Adamski J, Jakob F. Vitamin D signaling is modulated on multiple levels in health and disease. Mol Cell Endocrinol 2006;248:149–159. 7. Peng X, Hawthorne M, Vaishnav A, St-Arnaud R, Mehta RG. 25hydroxyvitamin D3 is a natural chemopreventive agent against carcinogen induced precancerous lesions in mouse mammary gland organ culture. Breast Cancer Res Treat 2009;113:31–41. 8. Peng X, Vaishnav A, Murillo G, Alimirah F, Torres KE, Mehta RG. Protection against cellular stress by 25-hydroxyvitamin D3 in breast epithelial cells. J Cell Biochem 2010;110:1324–1333. 9. Rennard SI, Wachenfeldt K. Rationale and emerging approaches for targeting lung repair and regeneration in the treatment of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2011;8:368–375. 10. Togo S, Holz O, Liu X, Sugiura H, Kamio K, Wang X, Kawasaki S, Ahn Y, Fredriksson K, Skold CM, et al. Lung fibroblast repair functions in patients with chronic obstructive pulmonary disease are altered by multiple mechanisms. Am J Respir Crit Care Med 2008;178:248–260. 11. Sato T, Liu X, Nelson A, Nakanishi M, Kanaji N, Wang X, Kim M, Li Y, Sun J, Michalski J, et al. Reduced miR-146a increases prostaglandin e(2)in chronic obstructive pulmonary disease fibroblasts. Am J Respir Crit Care Med 2010;182:1020–1029. 12. Dagouassat M, Gagliolo JM, Chrusciel S, Bourin MC, Duprez C, Caramelle P, Boyer L, Hue S, Stern JB, Validire P, et al. The cyclooxygenase-2-prostaglandin E2 pathway maintains senescence of chronic obstructive pulmonary disease fibroblasts. Am J Respir Crit Care Med 2013;187:703–714.

PGE2 release not only in fetal lung fibroblasts but also in adult lung fibroblasts. All three forms of vitamin D signal through the VDR and induce VDR nuclear translocation and stimulate the DNA-protein binding activity of vitamin D–responsive elements, and the VDR is required for the inhibitory effect of vitamin D on PGE2 release. Our findings that vitamin D modulates PGE2 synthesis and degradation by human lung fibroblasts suggest that vitamin D may regulate fibroblast-mediated lung tissue repair and remodeling. This provides a potential mechanism whereby vitamin D could modify lung structure and function. n Author disclosures are available with the text of this article at www.atsjournals.org.

13. Stumm CL, Wettlaufer SH, Jancar S, Peters-Golden M. Airway remodeling in murine asthma correlates with a defect in PGE2 synthesis by lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2011;301:L636–L644. 14. Dackor RT, Cheng J, Voltz JW, Card JW, Ferguson CD, Garrett RC, Bradbury JA, DeGraff LM, Lih FB, Tomer KB, et al. Prostaglandin e(2) protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction. Am J Physiol Lung Cell Mol Physiol 2011;301: L645–L655. 15. Borok Z, Gillissen A, Buhl R, Hoyt RF, Hubbard RC, Ozaki T, Rennard SI, Crystal RG. Augmentation of functional prostaglandin E levels on the respiratory epithelial surface by aerosol administration of prostaglandin E. Am Rev Respir Dis 1991;144:1080–1084. 16. Wilborn J, Crofford LJ, Burdick MD, Kunkel SL, Strieter RM, PetersGolden M. Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin e2 and to express cyclooxygenase-2. J Clin Invest 1995;95:1861–1868. 17. Pilewski JM, Liu L, Henry AC, Knauer AV, Feghali-Bostwick CA. Insulinlike growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition. Am J Pathol 2005;166:399–407. 18. Liu X, Kohyama T, Wang H, Zhu YK, Wen FQ, Kim HJ, Romberger DJ, Rennard SI. Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells. Am J Physiol Lung Cell Mol Physiol 2002;282:L1049–L1056. 19. Kobayashi T, Liu X, Wen FQ, Fang Q, Abe S, Wang XQ, Hashimoto M, Shen L, Kawasaki S, Kim HJ, et al. Smad3 mediates tgf-beta1 induction of vegf production in lung fibroblasts. Biochem Biophys Res Commun 2005;327:393–398. 20. Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S. 25hydroxyvitamin D3 1alpha-hydroxylase and vitamin d synthesis. Science 1997;277:1827–1830. 21. Hewison M, Zehnder D, Chakraverty R, Adams JS. Vitamin D and barrier function: a novel role for extra-renal 1 alpha-hydroxylase. Mol Cell Endocrinol 2004;215:31–38. 22. Ginde AA, Mansbach JM, Camargo CA Jr. Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the third national health and nutrition examination survey. Arch Intern Med 2009;169:384–390. 23. Herr C, Greulich T, Koczulla RA, Meyer S, Zakharkina T, Branscheidt M, Eschmann R, Bals R. The role of vitamin D in pulmonary disease: COPD, asthma, infection, and cancer. Respir Res 2011;12:31. 24. Gilbert CR, Arum SM, Smith CM. Vitamin D deficiency and chronic lung disease. Can Respir J 2009;16:75–80.


49

ORIGINAL RESEARCH 25. Kunisaki KM, Niewoehner DE, Connett JE. Vitamin D levels and risk of acute exacerbations of chronic obstructive pulmonary disease: a prospective cohort study. Am J Respir Crit Care Med 2012;185: 286–290. 26. Greiling D, Thieroff-Ekerdt R. 1alpha,25-dihydroxyvitamin d3 rapidly inhibits fibroblast-induced collagen gel contraction. J Invest Dermatol 1996;106:1236–1241. 27. Ramirez AM, Wongtrakool C, Welch T, Steinmeyer A, Zugel U, Roman J. Vitamin D inhibition of pro-fibrotic effects of transforming growth factor beta1 in lung fibroblasts and epithelial cells. J Steroid Biochem Mol Biol 2010;118:142–150. 28. Jones G. Pharmacokinetics of vitamin D toxicity. Am J Clin Nutr 2008; 88:582S–586S. 29. Shephard RM, Deluca HF. Plasma concentrations of vitamin D3 and its metabolites in the rat as influenced by vitamin D3 or 25hydroxyvitamin D3 intakes. Arch Biochem Biophys 1980;202:43–53.

50

30. Krishnan AV, Moreno J, Nonn L, Swami S, Peehl DM, Feldman D. Calcitriol as a chemopreventive and therapeutic agent in prostate cancer: role of anti-inflammatory activity. J Bone Miner Res 2007;22:V74–V80. 31. Delgado-Martinez AD, Martinez ME, Carrascal MT, Rodriguez-Avial M, Munuera L. Effect of 25-oh-vitamin D on fracture healing in elderly rats. J Orthop Res 1998;16:650–653. 32. Barreto AM, Schwartz GG, Woodruff R, Cramer SD. 25-hydroxyvitamin D3, the prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic epithelial cells. Cancer Epidemiol Biomarkers Prev 2000;9:265–270. 33. Lou YR, Molnar F, Perakyla M, Qiao S, Kalueff AV, St-Arnaud R, Carlberg C, Tuohimaa P. 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand. J Steroid Biochem Mol Biol 2010;118:162–170. 34. Lou YR, Laaksi I, Syvala H, Blauer M, Tammela TL, Ylikomi T, Tuohimaa P. 25-hydroxyvitamin D3 is an active hormone in human primary prostatic stromal cells. FASEB J 2004;18:332–334.
