Tissue inhibitor of metalloproteinase-4 instigates apoptosis in transformed cardiac fibroblasts

Tumor cells become malignant, in part, because of their activation of matri x metalloproteinases (MMPs) and inactivation of tissue inhibitor of metallo proteinases (TIMPs). Myocardial tumors are rarely malignant. This raises th e possibility that the MMPs and TIMPs are differentially regulated in the h eart compared to other tissues. Therefore, we hypothesized that a tissue sp ecific tumor suppressor exists in the heart. To test this hypothesis we pre pared cardiac tissue extracts from normal (n = 4), ischemic cardiomypathic ([CM) [n = 5], and dilated cardiomyopathic (DCM) [n = 8] human heart end-st age explants. The level of cardiospecific TIMP-4 was determined by SDS-PAGE and Western-blot analysis. The results suggested reduced levels of TIMP-4 in ICM and DCM as compared to normal heart. TIMP-4 was purified by reverse phase HPLC and gelatin-sepharose affinity chromatography. Collagenase inhib itory activity of chromatographic peaks was determined using fluorescein-co njugated collagen as substrate and fluorescence spectroscopy. The activity of TIMP-4 (27 kDa) was characterized by reverse zymography. The role of TIM P-4 in cardiac Fibroblast cell migration was examined using Boyden chamber analysis. The results suggested that TIMP-4 inhibited cardiac fibroblast ce lls migration and collagen gel invasion. To test whether TIMP-4 induces apo ptosis, we cultured cardiac normal and polyomavirus transformed fibroblast cells in the presence and absence of TIMP-4. The number of cells were measu red and DNA laddering was determined. The results suggested that TIMP-4 con trolled normal cardiac fibroblast transformation and induced apoptosis in t ransformed cells. Cardiospecific TIMP-4 plays a significant role in regulat ing the normal cell phenotype. The reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse extracellular matrix degradation (r emodeling), cardiac hypertrophy and failure. This study suggests a possible protective role of TIMP-4 in other organs which are susceptible to maligna ncy, J. Cell. Biochem. 80:512-521, 2001. (C) 2001 Wiley-Liss, Inc.