Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.

Abstract

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, lamiiin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 jug per 106 cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.The extracellular matrix (ECM) of multicellular organisms plays an active role in the formation and maintenance of tissues. The meshwork of ECM macromolecules is deposited by resident cells and provides a substrate for cell adhesion and migration, as well as a permeability barrier in cell-cell communication. A number of processes in normal tissue maintenance [e.g., wound healing (1, 2), bone resorption (3), and uterine involution (4)] as well as certain pathologic processes [for example, rheumatoid arthritis (5, 6), epidermolysis bullosa (7,8), corneal (9, 10) and gingival disease (11)] require connective tissue remodeling, involving a guided degradation of the preexisting ECM. Secreted proteases, required for initiation of degradation of the proteinaceous components of the ECM, also play an important part in processes requiring cell movement, such as tumor invasion and metastasis (12, 13). These enzymes constitute a family of structurally related metalloendoproteases. Previously, we characterized the expression, properties, and primary structure of human fibroblast collagenase (14-16), an enzyme responsible for the initiation of interstitial collagen degradation. In this report, we present the characterization of human fibroblast stromel...

Synthesis and secretion of matrix‐degrading metalloproteases by human skeletal muscle satellite cells

“…3, lane 4). It is unclear whether these represent the proenzyme and activated forms of stromelysin or may represent stromelysin and interstitial collagenase, since the latter enzyme also migrates in this molecular weight range and has been reported to be detectable by casein zymography (Wilhelm et al, 1987). The existence of a high molecular weight caseinolytic activity in the serum required for maintenance of the cultures further complicated the interpretation of the casein zymography, in that it made it technically difficult to rule out the possibility that the lower molecular weight caseinolytic bands induced on PMA treatment might be derived by modification of the high molecular weight serum caseinolytic activity.…”

“…This is in the range reported for induction of MMP-9 secretion by phorbol esters in other cell types. Zymography on casein substrate gels can reveal proteolytic activity due to stromelysin (MMP-3) (Wilhelm et al, 1987;Chin et al, 1985;Werb et al, 1989). There was no detectable caseinolytic activity in control (Fig.…”

“…These include interstitial (type I) collagenase (or MMP-1) (Harris et al, 1984), 72 kDa gelatinasekype JV collagenase (MMP-8) (Collier et al, 1988), 92 kDa gelatinaseltype IV collagenase (MMP-9) (Wilhelm et al, 1989), and stromelysin-1 (MMP-3), which can degrade proteoglycans, fibronectin, laminin, gelatin, and certain of the collagens (Wilhelm et al, 1987;Murphy et al, 1987;Matrisian, 1992). The only neutral matrix-degrading protease presently known to be secreted by muscle cells is plasminogen activator (Miskin et al, 1978;Festoff et al, 1982;Quax et al, 1992).…”

Specific amino acid substitutions in human collagenase cause decreased autoproteolysis and reveal a requirement for a second zinc atom for catalytic activity

“…Under pathological conditions, MMPs are instrumental in tumour invasiveness during metastasis and cartilage destruction during arthritis. They represent members of a gene family which includes fibroblast collagenase [l], neutrophil collagenase [2], gelatinases [3,4], stromelysins I, II, III , and pump-l [5]. Sequence comparison and functional analysis of the MMPs has revealed the presence of individual domains within the protein which give the MMPs characteristic properties.…”

A 25 kDa α2‐microglobulin‐related protein is a component of the 125 kDa form of human gelatinase

“…Gelatinase degrades a number of matrix components, including elastin [1], cartilage proteoglycan [l], collagen types IV [2], V [3], XI [1] and type I gelatin.…”