Vitamin D3 inhibits expression and activities of matrix metalloproteinase-2 and -9 in human uterine fibroid cells

Abstract

Study question: Can biologically active vitamin D3 [1,25(OH)₂D3] regulate the expression and activity of matrix metalloproteinases (MMPs) in human uterine fibroid cells?

Summary answer: 1,25(OH)₂D3 effectively reduced the expression and activities of MMP-2 and MMP-9 in cultured human uterine fibroid cells.

What is known already: Uterine fibroids (leiomyoma) express higher levels of MMP activity than adjacent normal myometrium, and this is associated with uterine fibroid pathogenesis. However, it is unknown whether 1,25(OH)₂D3 can regulate the expression and activities of MMPs in human uterine fibroid cells.

Study design, size, duration: Surgically removed fresh fibroid tissue was used to generate primary uterine fibroid cells.

Participants/materials, setting, methods: An immortalized human uterine fibroid cell line (HuLM) and/or primary human uterine fibroid cells isolated from fresh fibroid tissue were used to examine the expression of several MMPs, tissue inhibitors of metalloproteinases (TIMP) 1 and 2 and the activities of MMP-2 and MMP-9 after 1,25(OH)₂D3 treatment. Real-time PCR and western blots analyses were used to measure mRNA and protein expression of MMPs, respectively. Supernatant cell culture media were analyzed for MMP-2 and MMP-9 activities using a gelatin zymography assay.

Main results and the role of chance: 1-1000 nM 1,25(OH)₂D3 significantly reduced mRNA levels of MMP-2 and MMP-9 in HuLM cells in a concentration-dependent manner (P < 0.5 to P < 0.001). The mRNA levels of MMP-1, MMP-3, MMP-13 and MMP-14 in HuLM cells were also reduced by 1,25(OH)₂D3. 1,25(OH)₂D3 significantly reduced MMP-2 and MMP-9 protein levels in a concentration-dependent manner in both HuLM and primary uterine fibroid cells (P < 0.05 to P < 0.001). Moreover, 1,25(OH)₂D3 increased the mRNA levels of vitamin D receptor (VDR) and TIMP-2 in a concentration-dependent manner in HuLM cells (P < 0.05 to P < 0.01). 1,25(OH)₂D3 also significantly increased protein levels of VDR and TIMP-2 in all cell types tested (P < 0.05 to P < 0.001). Gelatin zymography revealed that pro-MMP-2, active MMP-2 and pro-MMP-9 were down-regulated by 1,25(OH)₂D3 in a concentration-dependent manner; however, the active MMP-9 was undetectable.

Limitations, reasons for caution: This study was performed using in vitro uterine fibroid cell cultures and the results were extrapolated to in vivo situation of uterine fibroids. Moreover, in this study the interaction of vitamin D3 with other regulators such as steroid hormone receptors was not explored.

Wider implications of the findings: This study reveals an important biological function of 1,25(OH)₂D3 in the regulation of expression and activities of MMP-2 and MMP-9. Thus, 1,25(OH)₂D3 might be a potential effective, safe non-surgical treatment option for human uterine fibroids.

Keywords: MMPs; TIMP-2; VDR; fibroids; vitamin D3.

Figures

Figure 1

Effect of 1,25(OH)2D3 on mRNA expression of MMPs in cultured immortalized human uterine fibroid (HuLM) cells. Total RNA was isolated from HuLM cells treated with increasing concentrations of 1,25(OH)2D3 (0, 1, 10, 100 and 1000 nM) for 48 h. Equal amounts of each total RNA was used to perform quantitative real-time PCR analyses as indicated. Total RNA (1 µg) was reverse transcribed to cDNA and then real-time PCR analyses were performed for MMP-2 (A) and MMP-9 (B), MMP-1 (C), MMP-3 (D), MMP-13 (E) and MMP-14 (F) using gene-specific forward and reverse primers as described in the section Materials and Methods. The mRNA expression levels of above MMPs were normalized with GAPDH (internal control) and the normalized values were used to generate the graphs. Data are means ± SEM, *P < 0.05, **P < 0.01 and ***P < 0.001 when compared with corresponding untreated control (0). §§P < 0.01 when compared between 1,25(OH)2D3-treated data points. These experiments were repeated twice with similar results.

Figure 2

Effect of 1,25(OH)2D3 on mRNA expressions of the VDR and TIMP 1 and 2 in cultured immortalized human uterine fibroid (HuLM) cells. Real-time PCR analyses for mRNA expressions of VDR, TIMP-1 and TIMP-2 were performed using gene-specific sense and anti-sense primers as described in the section Materials and methods. The mRNA expression levels of VDR, TIMP-1 and TIMP-2 were normalized with GAPDH and the normalized values were used to generate the graphs. Data are means ± SEM, n = 3, **P < 0.01 when compared with corresponding untreated control. §P < 0.05 when compared between 1,25(OH)2D3-treated data points. This experiment was repeated twice with similar results.

Figure 3

Effect of 1,25(OH)2D3 on protein expression of MMP 2 and 9 in cultured human uterine fibroid cells. Immortalized human uterine fibroid (HuLM) cells (A) and primary uterine fibroid cells (B) were serum starved and treated with increasing concentrations of 1,25(OH)2D3 (0, 1, 10, 100 and 1000 nM) for 48 h. For each condition, an equal amount of protein lysate was analyzed by western blotting with anti-MMP-2 and anti-MMP-9 antibodies. Western blot with anti-β-actin antibody was used as the loading control. The intensity of each protein band was quantified and normalized with corresponding β-actin, and the normalized values were used to generate the graphs. Data are the mean ± SEM, n = 3, *P < 0.05, **P < 0.01 and ***P < 0.001 when compared with corresponding untreated control. §§P < 0.01 and §§§P < 0.001 when compared between 1,25(OH)2D3-treated data points. This experiment was repeated twice with similar results.

Figure 4

Effect of 1,25(OH)2D3 on protein expression of VDR in cultured human uterine fibroid cells. Immortalized human uterine fibroid (HuLM) cells (A) and primary human uterine fibroid cells (B) were serum starved and treated with increasing concentrations of 1,25(OH)2D3 (0, 1, 10, 100 and 1000 nM) for 48 h. Equal amounts of each cell lysate were analyzed by western blotting using anti-VDR antibody. The intensity of each protein band was quantified and normalized with corresponding β-actin, and the normalized values were used to generate the graphs. *P < 0.05 and ***P < 0.001 when compared with corresponding untreated control. §§§P < 0.001 when compared between 1,25(OH)2D3-treated data points. This experiment was repeated twice with similar results.

Figure 5

Effect of 1,25(OH)2D3 on protein expression of TIMP-2 in cultured human uterine fibroid cells. Lysates from immortalized human uterine fibroid (HuLM) cells (A) and primary uterine fibroid cells (B) treated with increasing concentrations of 1,25(OH)2D3 (as described above) were analyzed by western blots using anti-TIMP-2 antibody. Western blot with the anti-β-actin antibody was used as the loading control. The intensity of each protein band was quantified and normalized with corresponding β-actin, and the normalized values were used to generate the graphs. Data are mean ± SEM, n = 3, *P < 0.05, **P < 0.01 and ***P < 0.001 when compared with corresponding untreated control. This experiment was repeated twice with similar results.

Figure 6

Effect of 1,25(OH)2D3 on gelatinolytic activities of MMP 2 and 9 in cultured human uterine fibroid cells. Both immortalized human uterine fibroid (HuLM) cells (A) and primary uterine fibroid cells (B) were serum starved and treated with increasing concentrations of 1,25(OH)2D3 (0, 1, 10, 100 M and 1000 nM) for 48 h. Each supernatant medium (40 µl/each sample) was analyzed in 7.5% SDS PAGE gelatin zymography as described in the section Materials and methods. The digestion of gelatin in the gel by MMP-2 and MMP-9 enzymes cause the negative (unstained) bands. The pro-MMP-2 (72 kDa) and active MMP-2 (65 kDa) digested bands were detected in the gels which were decreased by 1,25(OH)2D3. Similarly, the pro-MMP-9 (92 kDa) was also detected in supernatant media in both cell types, which were also decreased by 1,25(OH)2D3. The active form of MMP-9 was undetectable in supernatant media of both cell types. This experiment was repeated twice with similar results.