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

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.

Keywords: Fibroblasts; Matrix Metalloproteinase 9; Vitamin D.

Figure 1

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

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

(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.