Re: 비타민 D의 fibroblast 과증식에 의한 섬유화 방지 기전(폐)

[문형철]

Tuberc Respir Dis (Seoul)

. 2014 Aug 29;77(2):73–80. doi: 10.4046/trd.2014.77.2.73

Vitamin D Inhibits Expression‎ and Activity of Matrix Metalloproteinase in Human Lung Fibroblasts (HFL-1) Cells

Seo Hwa Kim 1, Moon Seong Baek 1, Dong Sik Yoon 1, Jong Seol Park 1, Byoung Wook Yoon 1, Byoung Su Oh 1, Jinkyeong Park 1, Hui Jung Kim 1,✉

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PMCID: PMC4165663  PMID: 25237378

Abstract

Background

Low levels of serum vitamin D is associated with several lung diseases. The production and activation of matrix metalloproteinases (MMPs) may play an important role in the pathogenesis of emphysema. The aim of the current study therefore is to investigate if vitamin D modulates the expression‎ and activation of MMP-2 and MMP-9 in human lung fibroblasts (HFL-1) cells.

Methods

HFL-1 cells were cast into three-dimensional collagen gels and stimulated with or without interleukin-1β (IL-1β) in the presence or absence of 100 nM 25-hydroxyvitamin D (25(OH)D) or 1,25-dihydroxyvitamin D (1,25(OH)2D) for 48 hours. Trypsin was then added into the culture medium in order to activate MMPs. To investigate the activity of MMP-2 and MMP-9, gelatin zymography was performed. The expression‎ of the tissue inhibitor of metalloproteinase (TIMP-1, TIMP-2) was measured by enzyme-linked immunosorbent assay. Expression‎ of MMP-9 mRNA and TIMP-1, TIMP-2 mRNA was quantified by real time reverse transcription polymerase chain reaction.

Results

IL-1β significantly stimulated MMP-9 production and mRNA expression‎. Trypsin converted latent MMP-2 and MMP-9 into their active forms of MMP-2 (66 kDa) and MMP-9 (82 kDa) within 24 hours. This conversion was significantly inhibited by 25(OH)D (100 nM) and 1,25(OH)2D (100 nM). The expression‎ of MMP-9 mRNA was also significantly inhibited by 25(OH)D and 1,25(OH)2D.

Conclusion

Vitamin D, 25(OH)D, and 1,25(OH)2D play a role in regulating human lung fibroblast functions in wound repair and tissue remodeling through not only inhibiting IL-1β stimulated MMP-9 production and conversion to its active form but also inhibiting IL-1β inhibition on TIMP-1 and TIMP-2 production.

배경

혈청 비타민 D 수치가 낮으면

여러 폐 질환과 연관된다.

매트릭스 메탈로프로테이나제(MMPs)의 생성 및 활성화는

폐기종의 병인에서 중요한 역할을 할 수 있다.

따라서 본 연구의 목적은

비타민 D가 인간 폐 섬유아세포(HFL-1) 세포에서

MMP-2 및 MMP-9의 발현과 활성화를 조절하는지 조사하는 것이다.

방법

HFL-1 세포를 3차원 콜라겐 젤에 주입하고,

100 nM 25-하이드록시비타민 D(25(OH)D) 또는

1,25-디하이드록시비타민 D(1,25(OH)2D)의 존재 여부와 관계없이

인터루킨-1β(IL-1β)로 48시간 동안 자극하였다.

그런 다음

MMP를 활성화하기 위해 배양 배지에 트립신을 첨가했다.

MMP-2 및 MMP-9의 활성을 조사하기 위해

젤라틴 자이모그래피를 실시했다.

조직 금속단백분해효소 억제제(TIMP-1, TIMP-2)의 발현은 효

소결합 면역흡착 분석법으로 측정하였다.

MMP-9 mRNA 및 TIMP-1, TIMP-2 mRNA 발현은

실시간 역전사 중합효소 연쇄반응으로 정량화하였다.

결과

IL-1β는

MMP-9 생산과 mRNA 발현을 유의하게 자극했다.

트립신은

잠재적 MMP-2와 MMP-9를 24시간 이내에 활성 형태인

MMP-2(66 kDa)와 MMP-9(82 kDa)로 전환시켰다.

이 전환은 25(OH)D(100 nM)와 1,25(OH)2D(100 nM)에 의해

유의하게 억제되었다.

MMP-9 mRNA 발현 또한

25(OH)D 및 1,25(OH)2D에 의해 유의하게 억제되었다.

결론

비타민 D, 25(OH)D 및 1,25(OH)2D는

IL-1β에 의해 자극된 MMP-9의 생성과 활성 형태로의 전환을 억제할 뿐만 아니라,

IL-1β가 TIMP-1 및 TIMP-2 생성에 미치는 억제 효과도 억제함으로써,

상처 치유 및 조직 재형성 과정에서

인간 폐 섬유모세포의 기능을 조절하는 역할을 합니다.

Keywords: Vitamin D, Matrix Metalloproteinase 9, Fibroblasts

Introduction

The role of vitamin D in calcium and bone homeostasis is well described. In the last years, it has been recognized that in addition to this classical function, vitamin D modulates a variety of processes and regulatory systems including host defense, inflammation, immunity, and repair1,2.

Low levels of serum vitamin D is associated with impaired pulmonary function, increased incidence of inflammatory, infectious or neoplastic diseases. Several lung diseases, all inflammatory in nature, may be related to activities of vitamin D including asthma, chronic obstructive pulmonary disease (COPD) and cancer3,4,5. The exact mechanisms underlying these data are unknown, however, vitamin D appears to impact on the function of inflammatory and structural cells of lung.

The aim of the current study therefore is to investigate if vitamin D modulates expression‎ and activation of matrix metalloproteinase (MMP)-9 in human lung fibroblasts (HFL-1) cells, and study the functions of vitamin D on inflammatory and structural cells of lung.

서론

비타민 D가

칼슘 및 뼈의 항상성 유지에 미치는 역할은 잘 알려져 있습니다.

최근 몇 년 동안, 비타민 D는

이러한 고전적인 기능 외에도 숙주 방어, 염증, 면역 및 회복을 포함한

다양한 과정과 조절 시스템을 조절한다는 것이 인정되고 있다1,2.

혈청 비타민 D 수치가 낮으면

폐 기능 장애, 염증성, 감염성 또는 신생물성 질환의 발병률 증가와 관련이 있다.

천식, 만성 폐쇄성 폐 질환 (COPD) 및 암3,4,5 등이 포함됩니다.

이러한 데이터의 정확한 기전은 알려지지 않았으나,

비타민 D는 폐의 염증성 및 구조적 세포 기능에 영향을 미치는 것으로 보입니다.

따라서 본 연구의 목적은

비타민 D가 인간 폐 섬유아세포(HFL-1)에서

매트릭스 메탈로프로테이나제(MMP)-9의 발현 및 활성화를 조절하는지 조사하고,

폐의 염증성 및 구조적 세포에 대한 비타민 D의 기능을 연구하는 것입니다.

Materials and Methods1. Materials

25-Hydroxyvitamin D (25(OH)D) or 1,25-dihydroxyvitamin D (1,25(OH)2D) were purchased from TOCRIS Bioscience (Ellisville, MO, USA). Interleukin-1β (IL-1β) was purchased from R&D Systems (Minneapolis, MN, USA). Tissue culture supplements and medium were purchased from GIBCO BRL (Life Technologies, Grand Island, NY, USA). Fetal calf serum (FCS) was purchased from BioFluids (Rockville, MD, USA).

2. Cell culture

Human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in 100-mm tissue culture dishes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ, USA) with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 50 mg/mL of penicillin, 50 mg/mL of streptomycin, and 0.25 mg/mL of Fungizone. The fibroblast were passaged every 3-5 days. Subconfluent fibroblasts were trypsinized (trypsin-EDTA; 0.05% trypsin, and 0.53 mM EDTA-4 Na) and used for collagen gel culture. Fibroblasts used in these experiments were between cell passages 15 and 20.

To assess the effect of vitamin D on fibroblast release of MMPs and tissue inhibitors of metalloproteinases (TIMPs), HFL-1 cells were cast into collagen gels and released into medium. HFL-1 cells in gels then treated with 1 mL per well serum-free DMEM (SF-DMEM), vitamin D, 25(OH)D (100 nM), or 1,25(OH)2D (100 nM) and IL-1β. On day 2, 2 µg trypsin was added into 5 mL gel floating medium to activate MMPs. The gel size of third day was measured in 24 hours after adding trypsin on the cell, media were harvested for enzyme-linked immunosorbent assay (ELISA) (TIMPs) or gelatin zymography EIA (MMPs) as described below. Cells were trypsinized and counted with a Coulter Counter.

3. Preparation of collagen

Type I collagen was extracted from rat tail tendons by a previously published method6,7. Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were carefully removed. After repeated washes with Tris-buffered saline and 95% ethanol, type I collagen was extracted in 4 mM acetic acid at 4℃ for 24 hours. Protein concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution. Sodium dodecyl sulfate polyacrylamide gel electrophoresis routinely demonstrated no detectable proteins other than type I collagen.

4. Preparation of collagen gels

Collagen gels were prepared by mixing the appropriate amounts of rat tail tendon collagen, distilled water, 4× concentrated DMEM, and cell suspension so that the final mixture resulted in 0.75 mg/mL of collagen, 4.5×105 cells/mL, and a physiological ionic strength.

Fibroblasts were always added last to minimize damage during the preparation of the collagen gels. The mixture (0.5-mL aliquots) was cast into each well of 24-well tissue culture plates (Falcon, Franklin Lakes, NJ, USA). Gelation occurred in 20 minutes at room temperature, after which the gels were released and transferred to 60-mm tissue culture dishes containing 5 mL of SF-DMEM and cultured at 37℃ in 5% CO2 for 4-5 days. To demonstrate the effects of cytokines on collagen gel contraction and collagen degradation, cytokines (5 ng/mL of IL-1β), vitamin D, 25(OH)D (100 nM), or 1,25(OH)2D (100 nM) or a combination of both was added to the culture medium. Gel area was measured daily with an image analysis system (Optimax V, Burlington, MA, USA).

5. Hydroxyproline assay

Hydroxyproline, which is directly proportional to type I collagen content, was measured by spectrophotometric determination8,9. Briefly, the medium surrounding the gels was completely removed, and the gels were transferred to a glass tube (KIMAX; Fisher Scientific, St. Louis, MO, USA) with 2 mL of 6N HCl. O2 was removed by ventilation with N2 for 30 seconds. The gels were hydrolyzed at 110℃ for 12 hours. The samples were dried with a vacuum centrifuge and redissolved in distilled H2O before measurement. Hydroxy-proline in the samples was reacted with oxidant (1.4% chloramines T in acetate-citric acid buffer; Sigma, St. Louis, MO, USA) and Ehrlich's reagent (0.4% p-dimethylaminobenzaldehyde; Sigma) in 60% perchloric acid (Fisher Chemical, Fair Lawn, NJ, USA) at 65℃ for 25 minutes, and hydroxyproline content was determined by spectrophotometer at 550 nm.

6. Gelatinase activity assay

To investigate the activity of gelatinase, gelatin zymography was performed. The supernatant-conditioned media were concentrated 10-fold by lyophilization and dissolved in distilled water. Gelatin zymography was performed with a modification of a previously published procedure10,11. Samples were dissolved in 23 electrophoresis sample buffer (0.5 M Tris·HCl, pH 6.8, 10% sodium dodecyl sulfate, 0.1% bromphenol blue, and 20% glycerol) and heated for 5 minutes at 95℃. Forty microliters of each sample were then loaded into each lane, and electrophoresis was performed at 45 mA/gel. After electrophoresis, the gels were soaked with 2.5% (vol/vol) Triton X-100 and gently shaken at 20℃ for 30 minutes. After this, the gels were incubated in the metalloproteinase buffer (0.06 M Tris·HCl, pH 7.5, containing 5 mM CaCl2 and 1 mM ZnCl2) for 18 hours at 37℃. The gels were then stained with 0.4% (wt/vol) Coomassie blue and rapidly destained with 30% (vol/vol) methanol, and 10% (vol/vol) acetic acid.

7. ELISA

The amount of TIMP-1 and TIMP-2 in the cultures was determined by ELISA. Floating collagen gels containing fibroblasts were cultured with or without IL-1β, vitamin D, 25(OH)D (100 nM), or 1,25(OH)2D for 3 days. After incubation, the supernatants surrounding the gels were collected and the concentration of TIMP-1 and TIMP-2 in culture media was also determined by ELISA technique. Ninety-six-well ELISA plates were coated overnight at 4℃ with 100 µL of anti-human TIMP-1 or TIMP-2 antibodies (R&D Systems) diluted in Voler's buffer (pH 9.6). Plates were then washed three times in phosphate buffered saline (PBS) with 0.05% Tween 20 (pH 7.2-7.4) and 100 µL of recombinant human TIMP-1 standards (31.25-4,000 pg/mL) or TIMP-2 standards (15.62-2,000 pg/mL) were added in duplicate. Samples (diluted 1:100 in PBS for TIMP-1 and 1:20-1:50 for TIMP-2) were added in duplicate to individual wells and incubated at room temperature for 2 hours. After three washes, 100 µL of biotinylated anti-human TIMP-1 antibody (R&D Systems) or biotinylated anti human TIMP-2 (R&D Systems) diluted in PBS-Tween were added for 1 hour. After another three washes, 100 µL of horseradish peroxidase-avidin conjugate (Zymed, San Francisco, CA, USA), diluted 1:20,000 in PBS-Tween, were added and incubated for 1 hour in room temperature. After the final three washes, 200 µL of tetramethyl benzidine substrate were added, and color was developed for 30 minutes at room temperature. The reaction was stopped by adding 50 µL of stop solution (1 M H2SO4), and the degree of color generated was determined by measuring the optical density at 450 nm in a microplate reader (Bio-Rad, Hercules, CA, USA).

8. Real-time reverse transcription polymerase chain reaction (RT-PCR)

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

rRNA was simultaneously tested using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems). Data were normalized by the internal control and expressed as fold change versus ethanol treatment.

9. Statistical analysis

All data are expressed as mean±standard error of the mean (SEM). Statistical comparison of paired data was performed using Student's t-test, whereas multigroup data were analyzed by ANOVA followed by the Tukey's (one-way) or Bonferroni's (two-way) post-hoc analysis using PRISM4 software (GraphPad Prism, San Diego, CA, USA). p<0.05 was considered significant.

Results1. Effect of vitamin D on collagen gel contraction in the presence of IL-1β

Human lung fibroblasts (HFL-1) cells were cast into three-dimensional collagen gels and stimulated with or without IL-1β (0.5 ng/mL) in the presence or absence of 100 nM 25(OH)D or 1,25(OH)2D for 48 hours. Trypsin was then added into the culture medium in order to activate MMPs. Gel size was measured by an image analyzer. The gel size of third day was measured in 24 hours after adding trypsin on the cell. IL-1β induced collagen gel degradation completely (Figure 1), vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited collagen degradation by IL-1β+trypsin as determined by hydroxyproline (0.12±0.07 µg/gel of IL-1β+trypsin vs. 2.00±0.08 µg/gel of IL-1β+trypsin+25(OH)D vs. 1.55±0.35 µg/gel of IL-1β+trypsin+1,25(OH)D; p<0.01) (Figure 2).

Figure 1.

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Vitamin D inhibits collagen degradation. HFL-1 cells were cast into collagen gels and released into medium, as shown. On day 2, 2 µg trypsin was added into a 5 mL gel floating medium in order to activate matrix metalloproteinases. After 24 hours of adding trypsin, that is, on day 3, gel size was measured, as shown. Interleukin-1β (IL-1β) induced collagen gel degradation completely, and vitamin D, 25(OH)D, and 1,25(OH)2D inhibited collagen degradation. **p<0.01 compared to IL-1β alone by one-way ANOVA followed by Tukey test. SF-DMEM: serum-free Dulbecco's modified Eagle's medium.

Figure 2.

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Gels were harvested and HO-proline amount was quantified, as described. **p<0.01 compared to interleukin-1β (IL-1β) alone by two-way ANOVA followed by Bonferroni test. SF-DMEM: serum-free Dulbecco's modified Eagle's medium.

2. Effect of vitamin D on IL-1β induced production of MMP-9 and MMP-2 in the HFL-1 cells

Expression‎ and activity of MMP-2 and MMP-9 (92 kDa) were assessed by gelatin zymography with culture media of collagen gel of fibroblasts in each condition. IL-1β stimulates MMP-2 and MMP-9, trypsin converts latent form of MMP-2 and MMP-9 into active form within 24 hours, vitamin D, 25(OH)D, and 1,25(OH)2D inhibit MMP-2 and MMP-9 production in response to IL-1β, and also inhibits activation of MMPs in induced by trypsin (Figure 3).

Figure 3.

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Interleukin-1β (IL-1β) stimulates matrix metalloproteinase (MMP)-9 and MMP-2, and trypsin converts latent form of MMP-2 and -9 into active form. Vitamin D, 25(OH)D, and 1,25(OH)2D inhibit MMP-2 and MMP-9 production in response to IL-1β, and also inhibits activation of MMPs induced by trypsin. SF-DMEM: serum-free Dulbecco's modified Eagle's medium.

This conversion was significantly inhibited by 25(OH)D (100 nM) and 1,25(OH)2D (100 nM) (62.6±6.5% inhibition by 25(OH)D, and 67.7±9.1% inhibition by 1,25(OH)2D) (Figure 4).

Figure 4.

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Active matrix metalloproteinase (MMP)-9 (82 kDa) was determined by scanning densitometry. Interleukin-1β (IL-1β) stimulates MMP-9 and MMP-2, and trypsine converts latent form of MMP-2 and -9 into active form. Vitamin D, 25(OH)D, and 1,25(OH)2D inhibit the activation of MMP induced by trypsin.

We next examined if vitamin D could inhibit MMP-9 production in response to IL-1β using real-time RT-PCR. HFL-1 cells were treated with vitamin D in presence or absence IL-1β for 24 hours. Total RNA extracted with Trizol. IL-1β significantly stimulated MMP-9 mRNA expression‎ which was partially but significantly blocked by 25(OH)D and 1,25(OH)2D (3.70±0.67 fold vs. ethanol control of IL-1β vs. 1.76±0.31 fold vs. ethonol control of IL-1β+25(OH)D, 1.97±0.35 fold vs. ethanol control of IL-1β+1,25(OH)2D; p<0.05) (Figure 5).

Figure 5.

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HFL-1 cells were treated with interleukin-1β (IL-1β) or vitamin D for 24 hours. Total RNA extracted with Trizol. mRNA was measured by real-time reverse transcription polymerase chain reaction. *p<0.05 compared to IL-1β alone by two-way ANOVA followed by Bonferroni test. SF-DMEM: serum-free Dulbecco's modified Eagle's medium. MMP-9: matrix metalloproteinase-9.

3. Effect of vitamin D on TIMP-1 and TIMP-2 production in the presence of IL-1β from HLF-1 cells

Expression‎ of TIMP-1 and TIMP-2 were assessed by ELISA with culture media of collagen gel of fibroblasts in each condition. IL-1β inhibits TIMP-1 and TIMP-2 production, and vitamin D, 25(OH)D and 1,25(OH)2D significantly blocked IL-1β inhibition on TIMP-1 (161.50±6.51 ng/day/106 cells of IL-1β vs. 194.51±2.06 ng/day/106 cells of IL-1β+25(OH)D, 232.20±11.23 ng/day/106 cells of IL-1β+1,25(OH)2D; p<0.05) (Figure 6A), and TIMP-2 production (2.42±0.32 ng/day/106 cells of IL-1β vs 4.275±0.195 ng/day/106 cells of IL-1β+25(OH)D, 3.82±0.04 ng/day/106 cells of IL-1β+1,25(OH)2D, p<0.05) (Figure 6B).

Figure 6.

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Interleukin-1β (IL-1β) inhibits tissue inhibitor of metalloproteinase (TIMP)-1 (A) and TIMP-2 (B) production, and 25(OH)D and 1,25(OH)2D significantly block IL-1β inhibition on TIMP-1 (A) and TIMP-2 (B) production. *p<0.05, **p<0.01 compared to IL-1β alone by one way ANOVA followed by Tukey test. SF-DMEM: serum-free Dulbecco's modified Eagle's medium.

We next examined if vitamin D could inhibit TIMP-1 and TIMP-2 production in response to IL-1β using real-time RT-PCR. HFL-1 cells were treated with vitamin D in presence or absence IL-1β for 24 hours. Total RNA extracted with Trizol. IL-1β inhibits TIMP-1 and TIMP-2 mRNA expression‎, and vitamin D, 25(OH)D, and 1,25(OH)2D significantly blocked IL-1β inhibition on TIMP-1(0.59±0.04 fold vs. ethanol control of IL-1β vs. 1.705±0.21 fold vs. ethanol control of IL-1β+25(OH)D, 1.74±0.08 fold vs. ethanol control of IL-1β+1,25(OH)2D; p<0.01) (Figure 7A) and TIMP-2 mRNA expression‎ (0.48±0.08 fold vs. ethanol control of IL-1β vs. 2.76±0.37 fold vs. ethanol control of IL-1β+25(OH)D, 2.07±0.19 fold vs. ethanol control of IL-1β+1,25(OH)2D; p<0.01) (Figure 7B).

Figure 7.

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(A) TIMP-1 mRNA. (B) TIMP-2 mRNA: **p<0.01 compared to interleukin-1β (IL-1β) treatment by two-way ANOVA followed by Bonferroni test. SF-DMEM: serum-free Dulbecco's modified Eagle's medium.

결과1. IL-1β 존재 하에서 비타민 D가 콜라겐 겔 수축에 미치는 영향

인간 폐 섬유아세포(HFL-1)를 3차원 콜라겐 겔에 주입하고, 100 nM 25(OH) D 또는 1,25(OH)2D 유무에 따라 48시간 동안 자극했다. 이후 MMP 활성화를 위해 배지에 트립신을 첨가했다. 겔 크기는 영상 분석기로 측정했다. 세 번째 날의 겔 크기는 세포에 트립신을 첨가한 후 24시간 후에 측정했다. IL-1β는 콜라겐 겔 분해를 완전히 유도했다 (그림 1), 비타민 D, 25(OH)D 및 1,25(OH)2D는 하이드록시프롤린 측정 결과 IL-1β+트립신에 의한 콜라겐 분해를 유의하게 억제하였다(IL-1β+트립신: 0.12±0.07 µg/젤 vs. IL-1β+트립신+25 (OH)D vs. 1.55±0.35 µg/gel of IL-1β+trypsin+1,25(OH)D; p<0.01) (그림 2).

그림 1.

새 탭에서 열기

비타민 D는 콜라겐 분해를 억제한다. HFL-1 세포를 콜라겐 젤에 주입하고 그림과 같이 배지에 방출하였다. 2일째, 매트릭스 메탈로프로테이나제 활성화를 위해 5mL 젤 부유 배지에 트립신 2µg을 첨가하였다. 트립신 첨가 24시간 후, 즉 3일째에 그림과 같이 젤 크기를 측정하였다. 인터루킨-1β(IL-1β)는 콜라겐 젤 분해를 완전히 유도했으며, 비타민 D, 25(OH)D 및 1,25(OH)2D는 콜라겐 분해를 억제했습니다. **p<0.01, 일원 분산 분석(ANOVA) 후 Tukey 검정 결과 IL-1β 단독 대비. SF-DMEM: 무혈청 Dulbecco's modified Eagle's medium.

그림 2.

새 탭에서 열기

젤을 수확하고 HO-프롤린 양을 정량화하였다. **p<0.01, 인터루킨-1β(IL-1β) 단독 대비, 이원 분산분석(ANOVA) 후 본페로니 검정. SF-DMEM: 무혈청 Dulbecco's modified Eagle's medium.

2. 비타민 D가 HFL-1 세포에서 IL-1β에 의해 유도된 MMP-9 및 MMP-2 생산에 미치는 영향

각 조건에서 섬유아세포의 콜라겐 젤 배양액을 사용하여 젤라틴 자이모그래피로 MMP-2 및 MMP-9(92 kDa)의 발현과 활성을 평가했습니다. IL-1β는 MMP-2 및 MMP-9를 자극하고, 트립신은 24시간 이내에 MMP-2 및 MMP-9의 잠재형(latent form)을 활성형(active form)으로 전환하며, 비타민 D, 25(OH)D 및 1,25(OH)2D는 IL-1β에 대한 반응으로 MMP-2 및 MMP-9의 생성을 억제하고, 트립신에 의해 유도된 MMP의 활성화를 억제합니다(그림 3).

그림 3.

새 탭에서 열기

인터루킨-1β(IL-1β)는 매트릭스 메탈로프로테이나제(MMP)-9 및 MMP-2를 자극하고, 트립신은 MMP-2 및 -9의 잠재 형태를 활성 형태로 전환합니다. 비타민 D, 25(OH)D 및 1,25(OH)2D는 IL-1β에 대한 반응으로 MMP-2 및 MMP-9 생성을 억제하고, 트립신에 의해 유도된 MMP의 활성화도 억제합니다. SF-DMEM: 무혈청 덜베코 수정 이글 배지.

이 전환은 25(OH)D (100 nM) 및 1,25(OH)2D (100 nM)에 의해 현저하게 억제되었습니다 (25(OH)D에 의한 62.6±6.5% 억제, 1,25(OH)2D에 의한 67.7±9.1% 억제). (그림 4).

그림 4.

새 탭에서 열기

활성 매트릭스 메탈로프로테이나제(MMP)-9(82 kDa)는 스캐닝 농도계로 측정하였습니다. 인터루킨-1β(IL-1β)는 MMP-9 및 MMP-2를 자극하며, 트립신은 MMP-2와 -9의 잠재형(latent form)을 활성형(active form)으로 전환시킵니다. 비타민 D, 25(OH)D 및 1,25(OH)2D는 트립신에 의해 유도된 MMP 활성화를 억제합니다.

다음으로 실시간 RT-PCR을 사용하여 비타민 D가 IL-1β에 대한 반응으로 MMP-9 생성을 억제할 수 있는지 조사하였다. HFL-1 세포를 IL-1β 유무에 따라 비타민 D로 24시간 처리하였다. Trizol로 총 RNA를 추출하였다. IL-1β는 MMP-9 mRNA 발현을 유의하게 자극하였으며, 이는 25(OH) D 및 1,25(OH)2D에 의해 부분적이지만 유의하게 차단되었다(IL-1β 단독 대조군 대비 3.70±0.67배, IL-1β+25(OH)D 대조군 대비 1.76±0.31배, IL-1β+1,25(OH)2D 대조군 대비 1.97±0.35배; p<0.05) (그림 5).

그림 5.

새 탭에서 열기

HFL-1 세포를 인터루킨-1β(IL-1β) 또는 비타민 D로 24시간 처리하였다. Trizol로 총 RNA를 추출하였다. mRNA는 실시간 역전사 중합효소 연쇄반응(RT-PCR)으로 측정하였다. *p<0.05, IL-1β 단독 처리군 대비, 이원 분산분석(ANOVA) 후 Bonferroni 검정. SF-DMEM: 무혈청 Dulbecco's modified Eagle's medium. MMP-9: 매트릭스 메탈로프로테이나제-9.

3. HLF-1 세포에서 IL-1β 존재 하에 비타민 D가 TIMP-1 및 TIMP-2 생산에 미치는 영향

각 조건에서 섬유아세포의 콜라겐 젤 배양액을 사용하여 ELISA로 TIMP-1 및 TIMP-2 발현을 평가했습니다. IL-1β는 TIMP-1 및 TIMP-2 생성을 억제하며, 비타민 D, 25(OH)D 및 1,25(OH)2D는 IL-1β에 의한 TIMP-1 억제를 현저히 차단했습니다. (IL-1β 단독 처리군: 161.50±6.51 ng/일/106 세포, IL-1β+25(OH)D 처리군: 194.51±2.06 ng/일/106 세포, IL-1β+1,25(OH)2D 처리군: 232.20±11.23 ng/일/106 세포; p<0.05) (그림 6A), 그리고 TIMP-2 생산량 (IL-1β: 2.42±0.32 ng/일/106 세포 vs 4.275±0. 195 ng/일/106 세포의 IL-1β+25(OH)D, 3.82±0.04 ng/일/106 세포의 IL-1β+1,25(OH)2D, p<0.05) (그림 6B).

그림 6.

새 탭에서 열기

인터루킨-1β(IL-1β)는 금속단백분해효소 억제제(TIMP)-1(A) 및 TIMP-2(B) 생성을 억제하며, 25(OH)D와 1,25(OH)2D는 IL-1β에 의한 TIMP-1(A) 및 TIMP-2(B) 생성 억제를 유의하게 차단한다. *p<0.05, **일원 분산 분석(ANOVA) 후 Tukey 검정 결과, IL-1β 단독 처리군 대비 p<0.01. SF-DMEM: 무혈청 Dulbecco's modified Eagle's medium.

다음으로 실시간 RT-PCR을 사용하여 비타민 D가 IL-1β에 대한 반응으로 TIMP-1 및 TIMP-2 생성을 억제할 수 있는지 조사하였다. HFL-1 세포를 IL-1β 유무에 따라 비타민 D로 24시간 처리하였다. Trizol로 총 RNA를 추출하였다. IL-1β는 TIMP-1 및 TIMP-2 mRNA 발현을 억제하며, 비타민 D, 25(OH)D 및 1,25 (OH)2D는 IL-1β에 의한 TIMP-1 억제를 현저히 차단하였다(에탄올 대조군 대비 0.59±0.04 배 vs. IL-1β, 에탄올 대조군 대비 1.705±0.21 배 vs. IL-1β+25(OH)D, 에탄올 대조군 대비 1.74±0.08 배 vs. IL-1β+1,25 (OH)2D; p<0.01) (그림 7A) 및 TIMP-2 mRNA 발현(0.48±0.08 배 vs. 에탄올 대조군 IL-1β vs. 2.76±0.37 배 vs. 에탄올 대조군 IL-1β+25(OH)D, 2.07±0.19 배 vs. 에탄올 대조군 대비 IL-1β+1,25(OH)2D; p<0.01) (그림 7B).

그림 7.

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(A) TIMP-1 mRNA. (B) TIMP-2 mRNA: **p<0.01, 인터루킨-1β(IL-1β) 처리 대비, 이원 분산분석(ANOVA) 후 본페로니 검정. SF-DMEM: 무혈청 딜베코 수정 이글 배지.

Discussion

This study demonstrates vitamin D play a role in regulating human lung fibroblast functions in wound repair and tissue remodeling through inhibiting IL-1β stimulated the MMP-2 and MMP-9 production from fibroblasts and inhibiting conversion from latent to active form of MMP-2 and MMP-9. Consistently, vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited collagen degradation by IL-1β+trypsin as determined by hydroxyproline assay. Vitamin D, 25(OH)D, and 1,25(OH)2D also significantly blocked IL-1β inhibition on TIMP-1 and TIMP-2 production from fibroblasts.

Emphysema has been believed to develop when mediators of tissue injury exceed protective mechanisms within the lung. Evidence also supports the concept that tissue destruction represents a balance between tissue injury and tissue repair12. If these repair responses can restore normal tissue architecture, function can be preserved. Efforts at repair, however, may result in disruption of normal tissue. In COPD, both in the airways and in the alveolar structures, tissue dysfunction likely results from altered structure due to incompletely effective repair responses13. The net tissue destruction that characterizes emphysema represents an imbalance between tissue destruction and tissue repair processes, analogous to the protease-antiprotease balance. Therefore, the levels and activities of MMPs and TIMPs that are potentially involved in alveolar destruction (emphysema) and extracellular matrix remodeling.

A parallel regulation of IL-1β-induced expression‎ of MMPs and TIMPs was observed in human fibroblasts14 and rat mesangial cells15. An elevated level of IL-1 is one of the key mediators that greatly enhances the biosynthesis and secretion of precursors of these MMPs (pro-MMPs) and prostaglandin E2 from mesenchymal cells at inflammatory sites16. The promotion of wound healing and tissue degradation is considered to be in part due to the production of MMPs by cells stimulated with IL-117.

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 remodeling18,19.

The culture of fibroblasts in three-dimensional collagen gels has been utilized as an in vitro system to eval‎uate tissue repair and remodeling20,21. When cultured in three-dimensional gels composed of native type I collagen, fibroblasts orient themselves along the collagen fibers. Both fibroblast proliferation and protein production in three-dimensional collagen gel culture differ markedly from those in routine tissue culture conditions20. Through interactions that depend in part on α2β1-integrins, fibroblasts can exert a tensile force on the collagen fibers. If the gels are unrestrained, for example in floating gel culture, the fibroblasts cause the gels to contract. This contraction can be modified by a variety of exogenous agents, which can either stimulate or inhibit collagen gel contraction22,23.

A recent study showed that vitamin D deficiency is highly preval‎ent in COPD and correlates with variants in the vitamin D binding gene24. There are several factors that could account for vitamin D deficiency in COPD patients: poor diet, a reduced capacity of aging skin for vitamin D synthesis, reduced outdoor activity and therefore sun exposure, an increased catabolism by glucocorticoids, impaired activation because of renal dysfunction, and a lower storage capacity in muscles or fat due to wasting25. Many steps of the vitamin D pathway (intake, synthesis, storage, metabolism) can potentially be disturbed in COPD patients.

Vitamin D belongs to a steroid hormone superfamily of nuclear receptors that has pleiotropic protective effects on several diseases and disorders including asthma and COPD26,27,28. 1,25(OH)2D3 (1,25-dihydroxyvitamin D3) an active metabolite of vitamin D (which binds with nuclear receptor vitamin D receptor [VDR] and interacts other steroid hormone receptors), is a potent regulator of the immune response in Th1 cell-directed diseases29,30.

Vitamin D is a ligand for nuclear hormone VDR, and upon binding it modulates various cellular functions. Vitamin D or VDR deficiency would invoke lung inflammation and alteration in lung function by proteinase/antiproteinase imbalance. Deletion of VDR leads to premature emphysema/COPD by increased matrix metalloproteinases and lymphoid aggregates formation31.

Vitamin D also to attenuates tumor necrosis factor-α induced upregulation of MMP-9 in keratinocytes32. Vitamin D deficiency may lead to a reduced attenuation of MMP-9 activity resulting in enhanced degradation of lung parenchyma.

In the current study, we have demonstrated that the latent form of MMP-2 and MMP-9 produced in fibroblasts in three-dimensional collagen gels with response to cytokines. Trypsin have a clear effect in converting the MMP-2 and MMP-9 to lower molecular mass forms that corresponded to active MMP-2 and MMP-9. Active form of MMP-2 and MMP-9 from fibroblasts in collagen gel, cultured with IL-1β+trypsin, induced degradation collagen gel completely, however, vitamin D, 25(OH)D, and 1,25(OH)2D significantly inhibited collagen degradation by IL-1β+trypsin (Figure 1) as determined by hydroxyproline assay. Thus the current study supports the concept that cytokines such as IL-1β can induce the production of MMPs but that maximal collagen degradation is achieved only in the presence of an activator such as trypsin.

Vitamin D, 25(OH)D, and 1,25(OH)2D inhibit MMP-2 and MMP-9 production in response to IL-1β, and also inhibits activation of MMPs in induced by trypsin (Figures 3, 4) as a function of modulator of extracellular matrix homeostasis. Using real-time RT-PCR, this study also showed IL-1β significantly stimulated MMP-9 mRNA expression‎ which was partially but significantly blocked by vitamin D, 25(OH)D, and 1,25(OH)2D in real-time RT-PCR (Figure 5). IL-1β inhibits TIMP-1 and TIMP-2 production from fibroblasts and vitamin D, 25(OH)D, and 1,25(OH)2D also significantly blocked IL-1β inhibition on TIMP-1 and TIMP-2 production in ELISA assay (Figure 6). IL-1β inhibits TIMP-1 and TIMP-2 mRNA expression‎, and vitamin D, 25(OH)D, and 1,25(OH)2D significantly blocked IL-1β inhibition on TIMP-1 and TIMP-2 mRNA expression‎ (Figure 7).

In summary, we have demonstrated that vitamin D, 25(OH)D, and 1,25(OH)2D play a role in regulating human lung fibroblast functions in wound repair and tissue remodeling through not only inhibiting IL-1β stimulated MMP-2 and MMP-9 production and conversion from latent to active form of MMP-2 and MMP-9, but also inhibiting IL-1β inhibition on TIMP-1 and TIMP-2 production.

Our findings that vitamin D modulates cytokine induced MMP synthesis and collagen 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 related to inflammation such as COPD.

토론

본 연구는 비타민 D가

상처 치유 및 조직 재형성 과정에서

인간 폐 섬유아세포 기능을 조절하는 역할을 수행함을 입증한다.

이는 섬유아세포에서

IL-1β에 의해 자극된 MMP-2 및 MMP-9 생성을 억제하고,

MMP-2 및 MMP-9의 잠재형에서 활성형으로의 전환을 억제함으로써 이루어진다.

일관되게, 비타민 D, 25(OH)D 및 1,25(OH)2D는

하이드록시프롤린 분석을 통해 확인된 바와 같이

IL-1β+트립신에 의한 콜라겐 분해를 현저히 억제하였다.

비타민 D, 25(OH)D 및 1,25(OH)2D는 또한

섬유아세포에서 IL-1β에 의한 TIMP-1 및 TIMP-2 생성 억제를 현저히 차단하였다.

폐기종은

조직 손상 매개체가 폐 내 보호 기전을 초과할 때 발생한다고 알려져 있다.

조직 파괴는

조직 손상과 조직 복구 사이의 균형을 나타낸다는 개념도 증거로 뒷받침된다12.

이러한 복구 반응이 정상적인 조직 구조를 회복시킬 수 있다면

기능도 보존될 수 있다.

그러나 복구 노력은

정상 조직의 파괴로 이어질 수도 있다.

만성 폐쇄성 폐질환(COPD)에서는

기도 및 폐포 구조 모두에서, 불완전한 복구 반응으로 인한 구조 변화로 인해

조직 기능 장애가 발생할 가능성이 높다13.

기종(肺氣腫)을 특징짓는 순 조직 파괴는

조직 파괴와 조직 회복 과정 간의 불균형을 나타내며,

이는 프로테아제-항프로테아제 균형과 유사하다.

따라서

폐포 파괴(기종) 및 세포외 기질 재형성에 잠재적으로 관여하는

MMPs와 TIMPs의 수준 및 활성이 중요하다.

인간 섬유아세포14와 쥐 사구간 세포15에서 IL-1β에 의해 유도된 MMP 및 TIMP 발현의 병행적 조절이 관찰되었다. 상승된 IL-1 수준은 염증 부위에서 중간엽 세포로부터 이러한 MMP 전구체(프로-MMP) 및 프로스타글란딘 E2의 생합성과 분비를 크게 증진시키는 핵심 매개체 중 하나이다16. 상처 치유와 조직 분해 촉진은 부분적으로 IL-1에 의해 자극된 세포의 MMP 생산에 기인하는 것으로 간주됩니다17.

섬유아세포는 조직 수복 및 재형성에 중요한 역할을 하며, 이는 COPD와 천식의 핵심 특징입니다. 섬유아세포는 세포외 기질 성분을 생성 및 변형시키고, 조직 재구성의 자크린 또는 파크린 조절제로 작용하는 매개체를 방출함으로써 조직 수복을 조절한다18,19.

섬유아세포를 3차원 콜라겐 겔에서 배양하는 방법은 조직 수복 및 재구성을 평가하기 위한 in vitro 시스템으로 활용되어 왔다20,21. 원생 제1형 콜라겐으로 구성된 3차원 겔에서 배양될 때, 섬유아세포는 콜라겐 섬유를 따라 배열된다. 3차원 콜라겐 겔 배양에서의 섬유아세포 증식과 단백질 생산은 일반적인 조직 배양 조건과 현저히 다르다20. α2β1-인테그린에 부분적으로 의존하는 상호작용을 통해, 섬유아세포는 콜라겐 섬유에 인장력을 가할 수 있다. 예를 들어 부유 겔 배양과 같이 겔이 제약받지 않을 경우, 섬유아세포는 젤을 수축시킵니다. 이 수축은 다양한 외인성 물질에 의해 조절될 수 있으며, 이러한 물질들은 콜라겐 젤 수축을 촉진하거나 억제할 수 있습니다22,23.

최근 연구에 따르면 비타민 D 결핍은 만성 폐쇄성 폐질환(COPD)에서 매우 흔하며, 비타민 D 결합 유전자의 변이와 상관관계가 있습니다24. COPD 환자의 비타민 D 결핍을 설명할 수 있는 몇 가지 요인이 있습니다: 불충분한 식이, 노화된 피부의 비타민 D 합성 능력 감소, 야외 활동 및 햇빛 노출 감소, 글루코코르티코이드에 의한 증가된 이화작용, 신장 기능 장애로 인한 활성화 장애, 그리고 근육이나 지방에서의 저장 능력 저하(소모성 질환으로 인해)25. 비타민 D 대사 경로의 여러 단계(섭취, 합성, 저장, 대사)가 COPD 환자에서 잠재적으로 방해받을 수 있습니다.

비타민 D는 핵 수용체 스테로이드 호르몬 초가족에 속하며, 천식과 COPD를 포함한 여러 질환에 대해 다중 보호 효과를 가집니다26,27,28. 1,25 비타민 D의 활성 대사산물인 (OH)2D3 (1,25-디하이드록시비타민 D3)은 핵 수용체인 비타민 D 수용체(VDR)와 결합하고 다른 스테로이드 호르몬 수용체와 상호작용하며, Th1 세포가 관여하는 질환에서 면역 반응의 강력한 조절제 역할을 합니다29,30.

비타민 D는 핵 호르몬 VDR의 리간드이며, 결합 시 다양한 세포 기능을 조절합니다. 비타민 D 또는 VDR 결핍은 단백분해효소/항단백분해효소 불균형에 의한 폐 염증 및 폐 기능 변화를 유발한다. VDR 결손은 매트릭스 금속단백분해효소 증가 및 림프구 응집체 형성에 의해 조기 폐기종/만성폐쇄성폐질환(COPD)을 초래한다31.

비타민 D는 또한 각질세포에서 종양괴사인자-α에 의해 유도된 MMP-9의 상향조절을 억제한다32.

비타민 D 결핍은 MMP-9 활성 억제 감소로 이어져 폐 실질 조직의 분해를 촉진할 수 있습니다.

본 연구에서는 사이토카인 반응 하에 3차원 콜라겐 겔 내 섬유아세포에서 생성된 MMP-2 및 MMP-9의 잠재형(latent form)을 확인하였습니다. 트립신은 MMP-2 및 MMP-9를 활성형 MMP-2 및 MMP-9에 해당하는 저분자량 형태로 전환시키는 뚜렷한 효과를 나타냈습니다. 콜라겐 겔 내 섬유아세포에서 유래한 활성형 MMP-2 및 MMP-9는 IL-1β+트립신과 함께 배양 시 콜라겐 겔을 완전히 분해시켰으나, 비타민 D(25(OH)D) 및 1,25 (OH)2D는 하이드록시프롤린 분석을 통해 확인된 바와 같이 IL-1β+트립신에 의한 콜라겐 분해를 현저히 억제하였다(그림 1). 따라서 본 연구는 IL-1β와 같은 사이토카인이 MMP 생산을 유도할 수 있지만, 트립신과 같은 활성화제가 존재할 때만 최대의 콜라겐 분해가 달성된다는 개념을 뒷받침한다.

비타민 D, 25(OH)D 및 1,25(OH) 2D는 IL-1β에 대한 반응으로 MMP-2 및 MMP-9 생성을 억제할 뿐만 아니라, 트립신에 의해 유도된 MMP의 활성화를 억제하여(그림 3, 4) 세포외 기질 항상성 조절 기능으로 작용함을 보여주었다. 실시간 RT-PCR을 이용한 본 연구는 또한 IL-1β가 MMP-9 mRNA 발현을 유의하게 자극하는 것을 보여주었으며, 이는 비타민 D, 25(OH) D 및 1,25(OH)2D에 의해 부분적이지만 유의하게 차단됨을 보여주었다(그림 5). IL-1β는 섬유아세포에서 TIMP-1 및 TIMP-2 생성을 억제하며, 비타민 D, 25(OH)D 및 1,25(OH)2D 역시 ELISA 분석에서 TIMP-1 및 TIMP-2 생성에 대한 IL-1β의 억제를 유의하게 차단하였다(그림 6). IL-1β는 TIMP-1 및 TIMP-2 mRNA 발현을 억제하며, 비타민 D, 25(OH)D 및 1,25(OH)2D는 IL-1β에 의한 TIMP-1 및 TIMP-2 mRNA 발현 억제를 유의하게 차단하였다(그림 7).

요약하면,

우리는 비타민 D, 25(OH)D 및 1,25(OH)2D가

상처 치유 및 조직 재형성 과정에서

인간 폐 섬유아세포 기능을 조절하는 데 역할을 한다는 것을 입증했습니다.

이는 단순히 IL-1β에 의해 자극된 MMP-2 및 MMP-9 생산과

MMP-2 및 MMP-9의 잠재형에서 활성형으로의 전환을 억제할 뿐만 아니라,

IL-1β가 TIMP-1 및 TIMP-2 생산에 미치는 억제 효과 자체를 차단함으로써 이루어집니다.

비타민 D가

인간 폐 섬유아세포의 사이토카인 유도 MMP 합성 및 콜라겐 분해를 조절한다는 본 연구 결과는

비타민 D가 섬유아세포 매개 폐 조직 수복 및 재형성을 조절할 수 있음을 시사한다.

이는 비타민 D가

COPD와 같은 염증 관련 폐 구조 및 기능을 수정할 수 있는

잠재적 기전을 제공한다.

Acknowledgements

This paper was supported by Wonkwang University in 2012.

Footnotes

No potential conflict of interest relevant to this article was reported.

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