Cloning of a complementary dna for rabbit proactivator. a metalloproteinase that activates synovial cell collagenase, shares homology with stromelysin and transin, and is coordinately regulated with collagenase

Abstract

Rabbit proactivator is a neutral metalloproteinase that activates another metalloproteinase, procollagenase, and degrades noncollagenous matrix. We describe the construction of an activator complementary DNA (cDNA) clone, which is 1.9 kb, that selects a 2.1-kb messenger RNA (mRNA) in Northern blot hybridizations. Nucleic acid sequence studies of the activator cDNA indicate 1) that it encodes protein M , 53,881, 2) that this protein exhibits -80% homology with rat transin, an oncogene-induced protein with a previously unknown function, and 3) that, in the first 172 residues, it is virtually identical to the rabbit metalloproteinase, stromelysin. Homology between rabbit activator and human skin collagenase is approximately 50%. Activator and collagenase mRNA are coordinately regulated; untreated cultures of rabbit synovial fibroblasts produce low levels of each protein, but addition of phorbol myristate acetate ( W M ) results in an increase in mRNA for both proteins by 2.5-5 hours. Adding alltrans-retinoic acid (lo-%) or dexamethasone (lO-'M) to phorbol-stimulated cells coordinately suppresses both activator and collagenase mRNA. Our data suggest the existence of coordinately regulated metalloproteinases that are important in the modulation of connective tissue metabolism.The metalloproteinase, collagenase (EC 3.2.24.7), is the only neutral proteinase able to initiate degradation of the interstitial collagens, types I, 11, and I11 (for review, see refs. 1 and 2). As such, collagenase plays a crucial role in the degradation of collagen that occurs as a result of both normal physiologic and disease processes (3). To study mechanisms that control the synthesis of collagenase, we have developed a model system of cultured rabbit synovial fibroblasts. These cells are readily available from the synovium of the knees of young rabbits and grow well in culture. Of experimental value is the fact that they make only minimal amounts of collagenase unless stimulated with such agents as interleukin-I , crystals of monosodium urate monohydrate, or the tumor promoter, phorbol myristate acetate (PMA) (r18). Addition of an inducer results in an increase in collagenase messenger RNA (mRNA), which is followed by rapid synthesis and secretion of collagenase protein (6,9). Equally valuable is the fact that collagenase synthesis can be antagonized by glucocorticoids and by retinoids (10,ll). It is believed that both compounds act at the level of transcription to decrease collagenase mRNA and protein, but do not affect the half-life of collagenase mRNA (12). This system has thus allowed us to study both the induction and suppression of collagenase synthesis.Rabbit synovial fibroblast collagenase is synthesized and secreted as an inactive polypeptide, procollagenase ( M , 57,000), with a minor species of glycosylated enzyme (M, 61,000) (9,13). Activation of

Prostromelysin and procollagenase genes are differentially UP‐regulated in chondrocytes from the knees of rabbits with experimental osteoarthritis

“…This cDNA was then used as template in the PCR with the 2 respective synthetic primers. These primers were separated by -500 basepairs, in each case corresponding to positions 557-577 (5' primer) and positions 1091-1 11 1 (3' primer) for pro-MMP1 (31), and positions 661-681 (5' primer) and 1168-1 188 (3' primer) for pro-MMP3 (32).…”

“…To confirm the identity of cloned cDNAs, several clones from each were sequenced with the dideoxynucleotide chaintermination technique (33) using both the forward and the reverse M13 sequencing primers. The cDNAs were of the expected sequences (31,32).…”

Differential regulation of metalloprotease steady‐state mRNA levels by IL‐1 and FGF in rabbit articular chondrocytes

“…The specific mRNAs were anal>zed by either Northern blot or slot-blot by hydridizing for 18 h with oligonucleotid¢ probes for rabbit pro~'ollagenas¢ (nucleotid¢ sequence 842-886) [19] and proaetivator/stromelysin (877-921) [20]. The oligonueleotides were 5'-end labeled with '~"P using T4 polynueleotide kinase, and the signal intensity was standardized by probing the tilters with 5'-end labeled oligo(dT) [21].…”

“…The steady-state mRNA levels were determined by probing with oligonucleotide probes for prostromelysin and procollagenase [19,20]. Initially, we established whether the oligonucleotide probes recognized and hybridized to the appropriate metalloprotease mRNAs induced by IL-1 in rabbit articular chondrocytes.…”

In Situ Hybridization Studies of Stromelysin and Collagenase Messenger RNA Expression in Rheumatoid Synovium

“…Expression of the rat analog, transin, has been demonstrated in normal and transformed fibroblasts (9, lo), and its expression in tumors is related to the invasive phenotype (1 1). In addition, there is evidence that stromelysin may act as an activator of procollagenase in vitro (12,13).…”