Modulation of neutral matrix metalloproteinases of involuting rat mammary gland by different cations and glycosaminoglycans

The synthesis and regulation of the matrix metalloproteinases (MMPs) are im portant factors contributing to the involution of mammary gland. In order t o understand the role of these MMPs in involution and in remodeling of the mammary gland, the different MMPs (130K, 68K, and 60K gelatinases) were par tially purified by gel filtration and affinity chromatography over gelatin Sepharose and subjected to kinetic analysis. Comparative analysis of the di fferent gelatinases showed that the 130K that appears at the early involuta ry phase and the constitutive 68K enzyme are more specific for Col IV of th e basement membrane, while the inducible 60K that appeared at the later pha se of involution degraded Col I more efficiently. These neutral proteinases required Ca2+/Zn2+ for their activity and the analysis of cation dependenc e revealed that Ca2+ at 10 mM concentration acid above completely inhibited the enzyme. The 60K was active at very low concentration of Zn2+ (5 mu M); but at higher concentration of Zn2+ (2 mM), where the 68K and 130K were ac tive, the 60K gelatinase was inhibited, indicating a difference in the cati on dependence of these enzymes. Chondroitin sulfate A and chondroitin sulfa te C caused inhibition of the 130K, 68K, and 60K, while hyaluronic acid and heparin did not show any effect, suggesting that the chondroitin sulfate p roteoglycan that decorates collagen in the ECM can modulate the activity of the collagenases in vivo. These results suggest that the 130K gelatinase e xpressed during the early phase of involution degraded Col IV of the baseme nt membrane, making the 60K gelatinase formed at a later stage of involutio n more accessible to its preferred substrate (Col I of the underlying strom a), highlighting the role of these MMPs in mammary gland involution. (C) 19 99 Wiley-Liss, Inc.