Vitamin D attenuates myofibroblast differentiation and extracellular matrix accumulation in nasal polyp-derived fibroblasts through smad2/3 signaling pathway

To investigate the potential role of vitamin D (1,25(OH)2D3) in preventing the development of nasal polyps, we examined the effect of vitamin D on myofibroblast differentiation and extracellular matrix (ECM) production in TGF-β1-induced nasal polyp-derived fibroblasts (NPDFs) and elucidated the mechanisms underlying its inhibitory effect. 1,25(OH)2D3 significantly reduced expression levels of α-SMA, a myofibroblast marker, and fibronectin, a representative ECM component, in a dose-dependent manner in TGF-β1-induced NPDFs. 1,25(OH)2D3 suppressed activated Smad2/3 in time-course. Up-regulation of α-SMA, fibronectin and phosphorylation of Smad2/3 by TGF-β1 was unaffected by 1,25(OH)2D3 in NPDFs after vitamin D receptor-specific siRNA transfection. We confirmed that the Smad2/3-specific inhibitor SIS3 inactivated Smad2/3 and reduced α-SMA and fibronectin expression. Furthermore, acetylation of histone H3 was compromised by 1,25(OH)2D3, leading to inhibition of collagen 1A1, collagen 1A2 and α-SMA gene expression. Treatment with 1,25(OH)2D3 also significantly suppressed TGF-β1-enhanced contractility and motility in a contraction assay and Transwell migration assay. Finally, 1,25(OH)2D3 had a similar effect in ex vivo organ cultures of nasal polyps. Taken together, our results suggest that 1,25(OH)2D3 might be an effective therapy for nasal polyps by reducing myofibroblast differentiation and ECM production mediated by Smad2/3-dependent TGF-β1 signaling pathways in NPDFs.

Vitamin D is synthesized in the skin or consumed via nutritional sources and modulates bone development and calcium homeostasis 6 . However, recent reports have shown that vitamin D also has a wide range of antifibrotic properties, including anti-inflammation, anti-proliferation, anti-apoptosis, and anti-epithelial-mesenchymal transition properties [7][8][9][10] . Several documents have shown that vitamin D deficiency is associated with the severity of asthma and the severity of bone erosion due to immune dysfunction in CRSwNP 11,12 . Vitamin D taken during pregnancy may be adversely linked to increased risk of asthma and allergic rhinitis in childhood 13 . In addition, vitamin D derivatives were shown to inhibit matrix metalloproteinase (MMP)-2 and MMP-9 as well as eotaxin and regulated on activation, normal T cell expressed and secreted (RANTES) secretions in nasal polyp-derived fibroblasts (NPDFs) from Taiwanese patients with CRSwNP 14,15 .
Little is known regarding the mechanisms involved in the anti-tissue remodeling effect of vitamin D in TGF-β1-induced NPDFs. TGF-β1-mediated activation of Smad signaling is responsible for tissue fibrosis and remodeling in several organs 16,17 . In this study, we investigated whether vitamin D could prevent myofibroblast differentiation and extracellular matrix synthesis in NPDFs and in ex vivo organ culture of nasal polyps. Furthermore, we investigated the potential mechanisms involved in the effects of vitamin D for the treatment of nasal polyps.

1,25(OH) 2 D 3 suppresses myofibroblast differentiation in nasal polyp-derived fibroblasts.
To examine the effects of vitamin D on myofibroblast differentiation and ECM production in NPDFs, we established NPDFs from patients with nasal polyps. The purity of NPDFs was confirmed based on spindle-shaped cell morphology under microscopic observation and by staining for vimentin, Thy-1, and E-cadherin, which are used as fibroblast and epithelial markers (data not shown). The effect of 1,25(OH) 2 D 3 on viability in NPDFs was analyzed using MTT assay after treatment for 72 hours; no significant toxicity was observed in NPDFs treated with 1,25(OH) 2 D 3 in a dose-dependent manner up to 1,000 nM (Fig. S1). Since the maximal concentration of 1,25(OH) 2 D 3 achievable in nasal tissue is unknown, 100 nM was chosen as the optimal dose based on an in vitro study of vitamin D in fibroblasts 18 .
1,25(OH) 2 D 3 abrogates myofibroblast differentiation and collagen production via reducing phosphorylation of smad2/3 mediated by binding to vitamin D receptors in nasal polyp-derived fibroblasts. Smad2/3 signaling is a critical pathway induced by TGF-β1. Smad2/3 phosphorylation and translocation into the nucleus regulate α-SMA and fibronectin through binding to pro-fibrotic genes 20 . We investigated whether 1,25(OH) 2 D 3 could block the Smad2/3 signaling pathway in NPDFs. TGF-β1 treatment induced phosphorylation of smad2/3 from 15 minutes to 4 hours and 1,25(OH) 2 D 3 inhibited TGF-β1-induced phosphorylation after 2 hours (Fig. 3A). To determine whether this inhibition mechanism is mediated by the vitamin D receptor (VDR), we confirmed knockdown of VDR using siRNA (Fig. 3B) and the effects of 1,25(OH) 2 D 3 via Western blotting. The effects of 1,25(OH) 2 D 3 significantly inhibited the phosphorylation of Smad2/3 and upregulated α-SMA and fibronectin through the formation of complex with VDR in NPDFs (Fig. 3C). We also found that enhancement of α-SMA and fibronectin expression and overproduction of total soluble collagen by TGF-β1 was suppressed in NPDFs after direct treatment with SIS3, a Smad2/3-specific inhibitor ( Fig. 3D and E), similar to that of treatment with 1,25(OH) 2 D 3 . Confocal microscopy to identify translocation of p-Smad2/3 (Fig. 3F) displayed that 1,25(OH) 2 D 3 markedly blocked translocation of p-Smad2/3 from the cytoplasm to the nucleus. Taken together, these data suggest that 1,25(OH) 2 D 3 has anti-fibrotic activity via Smad2/3, which is a downstream molecule in the TGF-β1 signaling pathway in NPDFs.

1,25(OH) 2 D 3 inhibits TGF-β1-induced contractile activity and cell migration in nasal polyp-derived fibroblasts.
Myofibroblasts play a central role in repair of wound tissues through their capacity to produce strong contractile forces and recruit cell migration 22 . Thus, we examined whether 1,25(OH) 2 D 3 regulates contractile activity and migration of myofibroblast using collagen gel contraction and Transwell migration assays. TGF-β1 stimulated contraction of the collagen gel to 54.0 ± 12.1% of the initial area 24 hours after TGF-β1 stimulation, as previously described 23,24 , and 1,25(OH) 2 D 3 significantly inhibited TGF-β1-induced contraction of collagen gel by 92.3 ± 4.5% of the initial area (p < 0.05). In addition, migration activity of NPDFs via TGF-β1 stimulus was significantly reduced by 1,25(OH) 2 D 3 from 333.3 ± 76.4 to 143.3 ± 20.8 (p < 0.05) (Fig. 5A,B). These results suggest that 1,25(OH) 2 D 3 inhibits the functional activity of myofibroblasts by regulating decreased contractile and cell migration activities.

1,25(OH) 2 D 3 inhibits expression levels of α-SMA and fibronectin and total collagen production
in ex vivo organ culture of nasal polyps. To confirm the inhibitory effects of 1,25(OH) 2 D 3 on protein expression of α-SMA and fibronectin and collagen production in human tissues, we performed ex vivo organ culture of nasal polyps. 1,25(OH) 2 D 3 significantly inhibited expression of α-SMA and fibronectin and total collagen production in ex vivo organ culture of nasal polyps treated with TGF-β1 (Fig. 6). These results strongly suggest

Discussion
In this study, we evaluated the anti-tissue remodeling role and underlying mechanisms of vitamin D action in the formation of nasal polyps (Fig. 7). Our results demonstrated that treatment with vitamin D reduced expression of α-SMA and fibronectin and total collagen production and functionally suppressed collagen contraction and cell migration in TGF-β1-induced NPDFs and ex vivo organ culture of nasal polyps. Taken together, these results suggest that vitamin D inhibited significant fibrotic alterations associated with myofibroblast differentiation and excessive ECM production in NPDFs stimulated by TGF-β1 through Smad2/3 signaling pathways.
Nasal polyp formation is a difficult and recalcitrant condition, with an unclear etiology and frequent recurrence in clinical rhinology 25 . Many studies have demonstrated that TGF-β1 is the prime stimulator of fibroblast activation and that it can induce activation and differentiation of fibroblasts into myofibroblasts expressing α-SMA. TGF-β1 promotes high levels of ECM deposition, which can lead to airway tissue remodeling [26][27][28] . We previously demonstrated that both mRNA and protein expression levels of α-SMA and TGF-β1 were markedly higher in nasal polyp tissues than in normal inferior turbinate tissues, suggesting that tissue remodeling is involved in nasal polyp formation 23 .
Vitamin D, a secosteroid hormone, has recently attracted considerable attention due to its wide range of biological activities in several organs 6 . Wang et al. demonstrated significantly low serum levels of vitamin D in CRSwNP patients 29 . Furthermore, vitamin D provides significant protection against human nasal polyp formation by reducing the size of nasal polyps and relieving the symptoms and signs of nasal polyps 30 . Anti-fibrotic and tissue remodeling activities of active vitamin D counteract pro-fibrotic TGF-β1, inhibiting myofibroblast activation and suppressing α-SMA expression in renal interstitial fibroblasts and lung fibroblasts 7,18 . However, the role of vitamin D in the pathogenesis of nasal polyp formation remains largely unexplored. Specifically, it is unclear whether vitamin D affects the essential functions of myofibroblast differentiation and ECM production in NPDFs. We hypothesized that vitamin D influences pro-fibrotic processes in NPDFs and ex vivo organ culture of nasal polyps. In agreement with previous reports, we showed that TGF-β1 greatly stimulated α-SMA expression, a specific marker of myofibroblast differentiation, whereas vitamin D counteracted this effect at both the transcript and protein levels. Similarly, vitamin D-treated NPDFs displayed diminished TGF-β1-related expression of fibronectin and production of total collagen. Taken together, these results indicate that vitamin D suppresses myofibroblast differentiation and ECM production in NPDFs stimulated by TGF-β1.  TGF-β1 induces myofibroblast differentiation in part via the Smad2/3 signaling pathway 28,31 . The activated complex is phosphorylated and forms a heteromeric receptor complex with TGF-βRI after active TGF-β1 binds to TGF-βRII; it subsequently phosphorylates Smad2 and Smad3, which binds to Smad4, followed by translocation into the nucleus where the complex increases α-SMA gene transcription. A previous study showed that vitamin D supplementation suppresses renal fibrosis through stimulation of vitamin D receptor-mediated transcription, which inhibits TGF-β1-Smad signal transduction 32 . According to a recent report, pro-fibrotic gene expression is mediated by Smad translocation to the nucleus and chromatin remodeling under response of TGF-β1, 1,25(OH) 2 D 3 subsequently blocks acetylation of histone H3 in TGF-β1-induced hepatic stellate cells 21 . Moreover, we previously proposed that the TGF-β1/Smad2/3 signaling pathways are involved in myofibroblast differentiation and ECM production in NPDFs 23 . Thus, we also used Western blot analysis, ChIP-qPCR, confocal microscopy, and measurement of total collagen to explain that vitamin D is associated with the TGF-β/Smad signaling pathway in TGF-β1-induced NPDFs. Interestingly, we found that the phosphorylation and translocation of Smad2/3 were significantly decreased by treatment with vitamin D in TGF-β1-induced NPDFs. In addition, knockdown of VDR-NPDFs showed no effect on p-Smad2/3 and tissue remodeling-mediated protein expression. Furthermore, SIS3 (a Smad3-specific inhibitor) treatment in TGF-β1-induced NPDFs caused downregulation of α-SMA and fibronectin protein expression and collagen production, similar to the effect of vitamin D. We also determined that vitamin D reduced histone H3 hyperacetylation, further compromising transcription of COL1A1, COL1A2 and α-SMA genes. Taken together, these data suggest that vitamin D ameliorates fibroblast differentiation into myofibroblasts and ECM production via Smad2/3-mediated processes in NPDFs.
Activated fibroblasts play a critical role in the wound repair and scarring processes that trigger wound contraction at the site of damage, to which fibroblasts begin to migrate 22 . Kumar et al. described that the increased contraction and migration induced by TGF-β1 were significantly reduced by mitomycin-C in human nasal mucosal fibroblasts 33 . In the present study, we showed that treatment with vitamin D suppressed enhancement of cellular functions such as gel contraction and migration of NPDFs after treatment with TGF-β1, suggesting that vitamin D has therapeutic potential for nasal polyps.
Since an in vivo model of nasal polyps has not been established, previous reports used nasal ex vivo organ culture to study the physiology and pathology of nasal polyps, providing an accessible means to mimic in vivo conditions, including cell-to-cell contact, cell-to-matrix integrity, and maintenance of three-dimensional structures 34,35 . In our study, we confirmed the inhibitory effects of vitamin D in ex vivo organ culture of nasal polyp tissue, positively reducing the TGF-β1-mediated effects on expression of α-SMA and fibronectin and production of collagen.
Based on the current evidence, we propose a model that supports the anti-tissue remodeling activity of vitamin D via mediating suppression of TGF-β1/Smad2/3 signaling pathways and downregulation of histone H3 (Fig. 7). Herein we described a mechanism by which vitamin D preferentially acts as an anti-tissue remodeling agent under TGF-β1-triggered conditions; for example, under constitutively enhanced myofibroblast differentiation and production of ECM in NPDFs. In this model, vitamin D functions as a promising therapeutic agent by inhibiting the expression of α-SMA, eventually leading to suppression of ECM production. Vitamin D appears to have anti-tissue remodeling activities related to its modulation of myofibroblast differentiation and ECM production in NPDFs under TGF-β1 stimulation via blockade of TGF-β1-Smad2/3 signaling pathways and hyperacetylation of histone 3, which could contribute to the treatment and prevention of nasal polyps.

Materials and Methods
Nasal polyp-derived fibroblast culture and treatment. Eight patients with nasal polyps were recruited from the Department of Otorhinolaryngology, Korea University Medical Center, and nasal polyp tissues were obtained during surgical procedures. All patients were nonsmokers and had not been treated with oral or topical corticosteroids or antibiotics for at least 4 weeks before surgery. There were no known allergies, asthma, or aspirin sensitivities among the patients. This study was approved by the Korea University Medical Center Institutional Review Board. Written informed consent was obtained from all subjects, and this study was conducted according to the principles of the Declaration of Helsinki.
Isolation and confirmation of NPDFs were conducted as previously described 36 . Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 10,000 units/mL penicillin, and 10,000 μg/mL streptomycin (Invitrogen) at 37 °C in a 5% CO 2 incubator. NFDFs at the third to seven passages were used in the following experiments.

Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Total RNA
was extracted using the TRIzol RNA isolation protocol, and the first-strand cDNA was synthesized using 2 μg RNA in 20 μL of reaction buffer with MMVL reverse transcriptase (Promega, Madison, WI) media was quantified by the Sircol collagen assay. (F) Representative fluorescein immunocytochemistry for fibronectin (green) with nuclear DAPI (blue). Scale bar = 50 μm. All data are presented as mean ± SEM. Four primary cell lines from different donors were used. All experiments were performed in at least triplicate and repeated at least three times. *p < 0.05 vs. control, † p < 0.05, † † p < 0.01 vs. TGF-β1.
Chromatin immunoprecipitation assay. NPDFs were treated with TGF-β1 and/or 1,25(OH) 2 D 3 for 4 hours. Cells were harvested for the Chip assay from Upstate (EZ ChIP kit, Millipore Inc. Billerica, MA) according to the manufacturer's instructions. Briefly, after fixation, nuclei from NPDFs were isolated, lysed, sheared with sonication. Afterward, sheared DNA was incubated with a normal mouse immunoglobulin G (Upstate), mouse anti-Ace-H3 antibody (Cell Signaling). After immunoprecipitation, the cross-linked DNA was released, reversed, and then purified with the provided spin column. Col1A1, Col1A2 and α-SMA expression were examined by quantitative PCR, performed on Quantstudio3 (Applied Biosystems, Foster City, CA) using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) followed by initial denaturing step at 95 °C for 15 seconds, 50 cycles of denaturing at 95 °C for 5 seconds. and annealing at 60 °C for 30 seconds. The sequence of Col1A1, Col1A2 and α-SMA primers was provided in previous documents 20,22 . Enrichment of ChIP-DNA was defined as the ratio of the PCR product of ChIP DNA to the input DNA.
Collagen gel contraction assay. NPDFs (3 × 10 5 ) were mixed with reconstituted collagen solution consisting of eight volumes of rat-tail tendon collagen type I (BD Bioscience, Bedford, MA) to one volume of reconstituted buffer (260 mM NaHCO 3 , 200 mM HEPES, 50 mM NaOH) on ice. Then, 500 μl of the reconstituted (A) Contractile activity was assessed by collagen gel contraction assay. These pictures show the results of one experiment; the contraction area was measured using an Image J analyzer. (B) Cell migration was assessed by Transwell migration assay. These pictures show one experimental result; cell number was measured using an Image J analyzer. All data are presented as mean ± SEM. Four primary cell lines from different donors were used. All experiments were performed in at least triplicate and repeated at least three times. *p < 0.05 vs. control, † p < 0.01 vs. TGF-β1.
collagen mixture was placed in each well of a 24-well tissue culture plate and allowed to polymerize at 37 °C for 1 hour. After polymerization, the gels were gently transferred to six-well culture plates containing 1.5 mL serum-free-DMEM with TGF-β1 (5 ng/mL) and/or 1,25(OH) 2 D 3 (100 nM). The gels were then incubated at 37 °C in a 5% CO 2 atmosphere for 3 days. The area of each gel was measured using an Image J analyzer (NIH, Bethesda, MA). Data are expressed as the percentage of area compared with the initial gel area.
Cell migration assay. For transwell migration assays, NPDFs (1.5 × 10 4 ) were seeded onto Transwell chambers (Corning Life Sciences, MA) and cultured for 48 hours in DMEM containing 10% FBS, TGF-β1 (5 ng/mL), and/or 1,25(OH) 2 D 3 (100 nM). Cells on the upper surface of the membrane were removed using cotton swabs, and then the cells on the lower surface of the membrane were stained using Diff-Quik staining (Sysmex, Kobe, Japan). Images of stained cells from five selected views were captured under microscopy at 200x magnification (Olympus BX51; Olympus, Tokyo, Japan).
Ex vivo organ culture of nasal polyps. Ex vivo organ culture of nasal polyps was performed as described previously by Cho et al. 33 . Briefly, 2 to 3 mm 3 of nasal polyp tissues were washed 3 times with phosphate-buffered saline (PBS) and rinsed with culture medium composed of DMEM, 2% FBS, 100 U/mL penicillin (Invitrogen), 100 mg/mL streptomycin (Invitrogen), and 0.25 mg/mL fungizone. The rinsed tissue fragments were placed on a pre-hydrated 10 × 10 × 61-mm gelatin sponge (Spongostan, Johnson & Johnson, San Angelo, TX) with the mucosa side facing up and the submucosa side facing down. Tissue fragments were placed onto 6-well plates filled with 1.5 mL of culture medium per well such that the mucosa was above the liquid phase. Nasal polyps were stimulated with TGF-β1 (5 ng/mL) and/or 1,25(OH) 2 D 3 (100 nM) for 72 hours. The amount of total soluble collagen in culture media was quantified using the Sircol assay. All data are presented as mean ± SEM. Four nasal polyp specimens from different donors were used. All experiments were performed in at least triplicate and repeated at least three times. *p < 0.05 vs. control, † p < 0.01 vs. TGF-β1.
Statistical analysis. All data are presented as mean ± SEM. The statistical significance of differences between groups was assessed by one-way analysis of variance for factorial comparisons and by Tukey's multiple comparison tests for multiple comparisons. All experiments were performed in at least triplicate and repeated at least three times.