Inhibition by female sex hormones of collagen degradation by corneal fibroblasts

Abstract

Purpose: Corneal fibroblasts contribute to collagen remodeling in the corneal stroma in part by mediating collagen degradation. Given that corneal structure is influenced by sex hormone status, we examined the effects of sex hormones on collagen degradation by corneal fibroblasts.

Methods: Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without sex hormones including 17β-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA). Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression and activity of matrix metalloproteinases (MMPs) were evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-κB) inhibitor NF kappa B Inhibitor-alpha (IκB-α) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively.

Results: 17β-Estradiol and progesterone each inhibited interleukin (IL)-1β-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner, whereas testosterone and DHEA had no such effect. MMP expression and activation in corneal fibroblasts exposed to IL-1β were also inhibited by 17β-estradiol and progesterone. These female sex hormones did not affect cell proliferation or viability. Both 17β-estradiol and progesterone inhibited the IL-1β-induced phosphorylation of p38 MAPK without affecting that of the MAPKs extracellular Signal-regulated Kinase (ERK) or c-jun N-terminal kinase (JNK). 17β-Estradiol also inhibited the IL-1β-induced phosphorylation of IκB-α.

Conclusions: 17β-Estradiol and progesterone inhibited MMP expression and activity in IL-1β-stimulated corneal fibroblasts and thereby suppressed collagen degradation by these cells.

Figure 1

Concentration-dependent inhibition by 17β-estradiol and progesterone of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with (white bars) or without (gray bars) IL-1β (0.1 ng/ml) and in the presence of the indicated concentrations of 17β-estradiol or progesterone for 36 h, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means±SEM of quadruplicates from an experiment that was repeated three times with similar results. *p<0.05 (Dunnett’s test) versus the corresponding value for cells cultured without female sex hormones.

Figure 2

Lack of effect of DHEA or testosterone on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with (white bars) or without (gray bars) IL-1β (0.1 ng/ml) and in the presence of the indicated concentrations of DHEA or testosterone for 36 h, after which the amount of degraded collagen was determined. Data are means±SEM of quadruplicates from an experiment that was repeated three times with similar results.

Figure 3

Time course of the inhibitory effects of 17β-estradiol and progesterone on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured for the indicated times in collagen gels in the absence or presence of IL-1β (0.1 ng/ml) and either 17β-estradiol (10 μM) or progesterone (10 μM) as indicated. The amount of degraded collagen was then determined. Data are means±SEM of quadruplicates from an experiment that was repeated three times with similar results. *p<0.05 (Student’s t test) for cells incubated with IL-1β plus 17β-estradiol or progesterone versus the corresponding value for those incubated with IL-1β alone.

Figure 4

Effects of 17β-estradiol on MMP abundance and activity in culture supernatants of corneal fibroblasts stimulated with IL-1β. Cells were cultured in collagen gels in the presence of IL-1β (0.1 ng/ml) and the indicated concentrations of 17β-estradiol for 36 h, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or were analyzed by gelatin zymography (B). Bands corresponding to the pro and active forms of MMPs are indicated. Similar results were obtained in three separate experiments.

Figure 5

Effects of progesterone on MMP abundance and activity in culture supernatants of corneal fibroblasts stimulated with IL-1β. Cells were cultured in collagen gels in the presence of IL-1β (0.1 ng/ml) and the indicated concentrations of progesterone for 36 h, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or were analyzed by gelatin zymography (B). Similar results were obtained in three separate experiments.

Figure 6

Lack of effect of 17β-estradiol or progesterone on the proliferation of corneal fibroblasts. Cells were cultured for 24 h in MEM and in the absence (negative control) or presence of various concentrations of 17β-estradiol or progesterone or of 10% FBS (positive control). Cell proliferation was evaluated by measurement of BrdU incorporation with an enzyme-linked immunosorbent assay. Data are means±SEM of quadruplicates from an experiment that was repeated three times with similar results. *p<0.05 versus negative control (Dunnett’s test).

Figure 7

Lack of a cytotoxic effect of 17β-estradiol or progesterone on corneal fibroblasts. Cells were incubated for 24 h in MEM in the absence (negative control) or presence of various concentrations of 17β-estradiol or progesterone, after which the culture supernatants were assayed for LDH activity with a colorimetric assay and measurement of absorbance at 490 nm. The amount of LDH released from cells by a cell lysis solution was determined as a positive control. Data are means±SEM from three independent experiments. *p<0.05 versus negative control (Dunnett’s test).

Figure 8

Effects of 17β-estradiol on IL-1β–induced MAPK and IκB-α phosphorylation in corneal fibroblasts. Cells were incubated in the absence or presence of 17β-estradiol (100 μM) for 12 h and then in the additional absence or presence of IL-1β (0.1 ng/ml) for 30 min. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, JNK, p38 MAPK, or IκB-α. Data are representative of three independent experiments.

Figure 9

Effects of progesterone on IL-1β–induced MAPK and IκB-α phosphorylation in corneal fibroblasts. Cells were incubated in the absence or presence of progesterone (10 μM) for 12 h and then in the additional absence or presence of IL-1β (0.1 ng/ml) for 30 min. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, JNK, p38 MAPK, or IκB-α. Data are representative of three independent experiments.