Identification of aggrecanase activity in medium of cartilage culture.
Erosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10,000-fold. The enzyme cleaves at the aggrecanase site (Glu(373)-Ala(374)) but not at the MMP site (Asn(341)-Phe(342)) in the interglobular domain of the aggrecan. It also cleaves at Glu(1971)-Leu(1972), which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca(2+)-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase. (+info)
CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch.
A matrix metalloproteinase (MMP)-like gene was identified in mouse to contain a conserved MMP catalytic domain and an RRRR motif. It lacks a classic cysteine switch, but it possesses two novel motifs: a cysteine array (Cys-X(6)-Cys-X(8)-Cys-X(10)-Cys-X(3)-Cys-X(2)-Cys), and a novel Ig-fold. It is named CA-MMP after the distinct cysteine array motif, and little is known about its biochemical function. In an attempt to characterize CA-MMP activity, the full-length sequence was expressed in mammalian cells and its product found to be cell-associated without detectable secretion. In light of this unusual finding, a chimera combining the catalytic domain of CA-MMP with the prodomain of stromelysin-3 was constructed to express a fully active enzyme in mammalian cells. Purified CA-MMP catalytic domain expresses proteolytic activity against protein substrates in an MMP inhibitor sensitive fashion. Taken together, it is concluded that CA-MMP is an MMP with distinct structure, biochemical properties and evolutionary history that may define a new subclass of the MMP superfamily. (+info)
Human membrane type-4 matrix metalloproteinase (MT4-MMP) is encoded by a novel major transcript: isolation of complementary DNA clones for human and mouse mt4-mmp transcripts.
Five distinct membrane-type matrix metalloproteinases (MT-MMP) have been reported by cDNA cloning. However, the mt4-mmp gene product (MMP-17) has not been identified yet in spite of the cDNA isolation [Puente et al. (1996), Cancer Res. 56, 944-949]. In this study, we re-examined the transcripts for human mt4-mmp by 5' RACE and identified two types of transcripts. The minor one corresponded to the cDNA reported by Puente et al. and failed to express protein, and the other is the major transcript that has an extended open reading frame and expressed 67 and 71 kDa translation products. Thus, functional mt4-mmp has been identified for the first time. (+info)
Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.
The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9. (+info)
Triiodothyronine induces collagenase-3 and gelatinase B expression in murine osteoblasts.
Triiodothyronine (T3) increases bone resorption, but its effects on matrix metalloprotease (MMP) expression in bone are unknown. We tested the effects of T3 on collagenase-3 and gelatinase A and B expression in MC3T3 osteoblastic cells. T3 at 1 nM to 1 microM for 24-72 h increased collagenase-3 and gelatinase B mRNA levels, but it did not increase gelatinase A transcripts. In addition, T3 increased immunoreactive collagenase and gelatinase activity. Cycloheximide prevented the stimulatory effect of T3 on collagenase-3 but not on gelatinase B transcripts. Indomethacin did not prevent the effect of T3 on either MMP. T3 did not alter the decay of collagenase-3 or gelatinase B mRNA in transcriptionally arrested MC3T3 cells, and it increased the rate of collagenase-3 and gelatinase B gene transcription. Although T3 enhanced the expression of the tissue inhibitor of metalloproteinase-1 in MC3T3 cells, it increased collagen degradation in cultured intact rat calvariae. In conclusion, T3 increases collagenase-3 and gelatinase B synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the actions of T3 on bone matrix remodeling. (+info)
Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors.
We recently showed that retroviral vectors can be targeted through protease substrate interactions. Infectivity is blocked by a polypeptide fused to the viral envelope glycoprotein (SU) and is restored when a protease cleaves the connecting linker, releasing the inhibitory polypeptide from the viral surface. Protease specificity is achieved by engineering the sequence of the linker. Here, using two different matrix-metalloproteinase (MMP)-activatable vectors, we demonstrated highly efficient and selective transduction of MMP-rich target cells in a heterogeneous cell population. In vivo, the MMP-targeted vectors showed strong selectivity for MMP-rich tumor xenografts. Protease-activatable vectors offer new possibilities for in vivo targeting of gene delivery. (+info)
Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium.
Matrix metalloproteinases (MMPs) are zinc-requiring enzymes that can degrade components of the extracellular matrix and that are implicated in tissue remodeling. Their role in the onset of menstruation in vivo has been proven; however, the expression and functions of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in vascular structures are poorly understood. We determined by immunocytochemistry, using characterized monoclonal antibodies, the distribution of MMPs and of their inhibitors TIMP-1 and TIMP-2 in the endometrium during the menstrual cycle. MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 had differing distributions and patterns of expression. In addition to the localization of MMP-9 in the epithelium and of MMP-2, MMP-3, and MMP-1 in the stromal tissue, these MMPs were detected in the vascular structures. MMP-2 (72-kDa gelatinase) and tissue inhibitors TIMP-1 and TIMP-2 were detectable in vessels throughout the cycle. In contrast, MMP-3 (stromelysin-1) was detected only in late-secretory and menstrual endometrial vessels, while MMP-9 (92-kDa gelatinase) was detected in spiral arteries during the secretory phase and in vascular structures during the midfollicular and menstrual phases. The expression of MMP-2 and MMP-9 in endometrial vessels during the proliferative and secretory periods suggests their relationship to vascular growth and angiogenesis. The pronounced expression of MMP-3 (stromelysin-1) in the vessels situated in the superficial endometrial layer during menses suggests that this metalloproteinase initiates damage in the vascular wall during menstrual breakdown. The finding of an intense expression of TIMP-1 and TIMP-2 in the vessels delineating necrotic from non-necrotic areas during menses also suggests that they could limit tissue damage, allowing regeneration of the endometrium after menses. These data indicate that, in addition to expression in epithelial cells and stromal tissue, MMPs are expressed in endometrial vascular cells in a cycle-specific pattern, consistent with regulation by steroid hormones and with specific roles in the vascular remodeling processes occurring in the endometrium during the cycle. (+info)
Regulation of matrix metalloproteinases (MMPs) and their tissue inhibitors in human myometrial smooth muscle cells by TGF-beta1.
The objective of the present study was to determine whether transforming growth factor beta (TGF-beta) regulates the expression of matrix metalloproteinases (MMP) and the tissue inhibitor of MMP (TIMP) in myometrial smooth muscle cells. Using primary cultures of human myometrial smooth muscle cells we found that these cells express MMP-1, MMP-3, TIMP-1 and TIMP-2 mRNA and protein, with significantly higher values of TIMP than MMP. We also found that TGF-beta1 (1 ng/ml) increased the expression of TIMP-1 mRNA, while it reduced the expression of MMP-1 and MMP-3 mRNA, compared with untreated controls. In addition, TGF-beta1 slightly increased the production of TIMP-1, but not TIMP-2. Production of MMP-1 and MMP-3 was reduced by treatment with TGF-beta1, compared with the untreated control. A major portion of MMP-1 released into the culture-conditioned media was in complex with TIMP-1, and the levels of this complex were reduced by treatment with TGF-beta1. In conclusion, the data indicate that myometrial smooth muscle cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by TGF-beta1. Such a differential regulation of MMP and TIMP by TGF-beta may influence the rate of extracellular matrix (ECM) turnover following tissue injury, induced during myomectomy and Caesarean section, or in leiomyomas during growth. (+info)