Cytokines regulate matrix metalloproteinases and migration in cardiac fibroblasts

Abstract

We sought to define the relationship between cytokine stimulated release of matrix metalloproteinases (MMPs) and cell migration using adult rat cardiac fibroblasts. Interleukin-1beta (IL-1beta) increased release of MMP-2, -3, and -9, and TIMP-1, by 3-6-fold, measured by immunoblotting and gel zymography. Tumor necrosis factor-alpha (TNFalpha) augmented IL-1beta stimulated release of MMP-9, but not MMP-2 or -3. Transforming growth factor-beta1 (TGFbeta1) attenuated all the responses to IL-1beta. IL-1beta was also the most robust stimulus of adult rat cardiac fibroblast migration, measured in Boyden chamber assays. The combination of IL-1beta plus TNFalpha substantially enhanced migration, whereas TGFbeta1 strongly inhibited the migratory response to IL-1beta. The pan-selective MMP inhibitor GM 6001 effectively blocked IL-1beta stimulated migration. Pharmacologic inhibitors selective for ERK, JNK, and p38 MAP kinase pathways inhibited the IL-1beta regulation of individual MMPs. Increased MMP activity associated with migration of cardiac fibroblasts may be important determinants of cytokine-directed remodeling of injured myocardium.

Figure 1

IL-1β and TGFβ1 regulation of MMP activity and abundance. Cardiac fibroblast cultures were grown to confluence, stimulated with the indicated concentrations of cytokines, and supernatants analyzed for MMP activity by gel zymography, or enzyme abundance by immunoblotting with antibodies against specified MMPs, as described in Materials and Methods. Representative experiments are shown.

Figure 2

Interactions of IL-1β and TGFβ1 on regulation of MMP abundance. Cardiac fibroblasts were treated with indicated combinations of IL-1β and TGFβ1 and the culture supernatants were analyzed for MMP expression by immunoblotting with quantitative densitometry as described in Materials and Methods. Data represent mean ± SEM from 4–7 separate experiments for each treatment and are normalized relative to control (CON). *, p < 0.05 versus control; †, p< 0.05 versus IL-1β.

Figure 3

Cytokine regulation of cardiac fibroblast migration. Cardiac fibroblasts were treated with the indicated cytokines and assayed for cell migration as described in Materials and Methods. Data represent mean ± SEM from 3 separate experiments for each treatment and are normalized relative to the response to IL-1β. *, p < 0.05 versus control; †, p< 0.05 versus IL-1β.

Figure 4

Dependence of IL-1β stimulated MMP production on MAP kinase pathways. Cardiac fibroblasts were treated with IL-1β or no cytokine addition (CON) in the absence and presence of specified MAP kinase inhibitors. Production of MMP-2, 3, and 9 were quantitated by immunoblotting and densitometry as described in Materials and Methods. Data represent mean ± SEM from 4 separate experiments for each treatment and are normalized relative to control (CON). MEK-I, U01216, 10 μM; JNK-I, SP 600125, 10 μM; p38-I, SB 202190, 1 μM. *, p < 0.05 versus control; †, p < 0.05 versus IL-1β.