Age-dependent loss of MMP-3 in Hutchinson-Gilford progeria syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, progressive segmental premature aging disease that includes scleroderma-like skin, progressive joint contracture, and atherosclerosis. Affected individuals die prematurely of heart attacks or strokes. Extracellular matrix dysregulation is implicated as a factor in disease progression. We analyzed messenger RNA and protein levels for matrix metalloproteinases (MMPs)-2,-3, and -9 in HGPS primary human dermal fibroblasts using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. MMP-3 messenger RNA and protein levels decreased significantly with increasing donor age in HGPS fibroblasts but not in controls. MMP-2 messenger RNA also showed a donor age-dependent decrease in HGPS fibroblasts, but levels of secreted protein were unchanged. MMP-9 was similar in HGPS and control cultures. The decreased MMP-3 may represent a shift in the inherent extracellular matrix-degrading proteolytic balance in favor of matrix deposition in HGPS. This metalloproteinase has the potential to serve as a biomarker of therapeutic efficacy when assessing treatments for HGPS.

Figure 1.

Western blotting demonstrates that progerin protein is produced by all Hutchinson–Gilford progeria syndrome (HGPS) cell lines but not by control cell lines. Prelamin A, lamin A, and lamin C proteins are produced by all cell lines.

Figure 2.

Levels of messenger RNA for MMP genes were determined by real-time reverse-transcription polymerase chain reaction in five pairs of donor age–matched human dermal fibroblast lines. Symbols (○ = controls; ▲ = Hutchinson–Gilford progeria syndrome) indicate mean relative MMP-3 (A), -2 (B), and -9 (C) RNA levels standardized to β-actin for two separate experiments analyzed in duplicate; solid lines indicate the mixed linear model’s predicted mean with dashed lines representing upper and lower 95% confidence intervals.

Figure 3.

MMP-3 secretion (A and B) was determined in five donor age–matched pairs of human dermal fibroblast lines from 24-hour conditioned medium utilizing an enzyme-linked immunosorbent assay (n = 3 experiments per pair). (A) Bars (white = control; black = Hutchinson–Gilford progeria syndrome [HGPS]) indicate mean MMP-3 secretion of all five fibroblast lines per group, and error bars indicate upper and lower 95% confidence intervals. (B) Symbols (○ = controls; ▲= HGPS) indicate mean secreted protein levels from three replicate experiments for each individual cell line; solid lines indicate the mixed linear model’s predicted mean with dashed lines representing upper and lower 95% confidence intervals. Gelatin zymogram (C) of two representative samples of 24-hour conditioned media normalized to total cellular protein from control and HGPS cells. Enzyme levels of MMP-2 (D) and MMP-9 (E) were scored based on band intensity. Bars (white = control; black = HGPS) indicate means of all five fibroblast lines per group with standard errors (n = 2 experiments per pair).