A catalytically inactive gelatinase B/MMP-9 mutant impairs homing of chronic lymphocytic leukemia cells by altering migration regulatory pathways

We previously showed that MMP-9 overexpression impairs migration of primary CLL cells and MEC-1 cells transfected with MMP-9. To determine the contribution of non-proteolytic activities to this effect we generated MEC-1 transfectants stably expressing catalytically inactive MMP-9MutE (MMP-9MutE-cells). In xenograft models in mice, MMP-9MutE-cells showed impaired homing to spleen and bone marrow, compared to cells transfected with empty vector (Mock-cells). In vitro transendothelial and random migration of MMP-9MutE-cells were also reduced. Biochemical analyses indicated that RhoAGTPase and p-Akt were not downregulated by MMP-9MutE, at difference with MMP-9. However, MMP-9MutE-cells or primary cells incubated with MMP-9MutE had significantly reduced p-ERK and increased PTEN, accounting for the impaired migration. Our results emphasize the role of non-proteolytic MMP-9 functions contributing to CLL progression.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Induction of matrix metalloproteinase MMP-9 (92-kDa gelatinase) by retinoic acid in human neuroblastoma SKNBE cells: relevance to neuronal differentiation

Retinoic acid (RA) has been shown to induce human neuroblastoma SKNBE cell differentiation into a neuronal phenotype. Whether this neuronal differentiation is associated with modulation of matrix gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression was investigated in SKNBE cell cultures exposed to RA for 14 days. Their differentiation into a neuronal phenotype was typified by neural cell adhesion molecule and growth-associated protein-43 expression. Gelatinase expression was assessed by gel zymography, quantitative RT-PCR, and immunocytochemistry. Neuronal markers were located in neurites and ganglion-like clusters of neuronal cells induced upon RA exposure. MMP-2 expression was constitutive and remained unchanged at both the mRNA and protein levels in response to RA, tumor necrosis factor-alpha (TNFalpha), or phorbol 12-myristate 13-acetate (PMA) treatment. In contrast, MMP-9 was inducible by RA, TNFalpha, or PMA. MMP-9 was progressively enhanced by RA as a function of time exposure until day 14. The addition of TNFalpha or PMA potentiated RA-induced MMP-9 expression with a synergic maximal effect at day 14 of RA exposure. Immunoreactive MMP-9 was located early in outgrowing neurites, but only at day 14 of RA exposure in extensive neuritic networks. Taken together, the correlation between the MMP-9 expression by SKNBE cells and the time scale of their differentiation into a neuronal phenotype allowed us to propose that MMP-9 could participate in the neurite growth process and cell migration and organization into ganglion-like clusters.     

Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells

The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.     

Temporal-spatial co-localisation of tissue transglutaminase (Tgase2) and matrix metalloproteinase-9 (MMP-9) with SBA-positive micro-fibres in the embryonic kidney cortex

Growth of the kidney is a complex process piloted by the collecting duct (CD) ampullae. The dichotomous arborisation and consecutive elongation of this tubular element determines the exact site and time for the induction of nephrons in the overlaying mesenchymal cap condensates. The mechanism by which the CD ampullae find the correct orientation is currently unknown. Recently, we have demonstrated micro-fibres that originate from the basal aspect of the CD ampullae and extend through the mesenchyme to the organ capsule. The micro-fibres are assumed to be involved in the growth and arborisation process of the CD ampulla. Therefore, we have investigated the specific distribution of the micro-fibres during branching morphogenesis. We have also analysed whether the micro-fibres co-localise with extracellular matrix (ECM)-modulating enzymes and whether the co-localisation pattern changes during CD ampulla arborisation. Micro-fibres were detected in all stages of CD ampulla arborisation. Tissue transglutaminase (Tgase2) co-localised with soybean agglutinin (SBA)-positive micro-fibres, whose presence depended upon the degree of CD branching. Matrix metalloproteinase-9 (MMP-9) also co-localised with micro-fibres, but its expression pattern was different from that for Tgase2. Western blotting experiments demonstrated that Tgase2 and MMP-9 co-migrated with SBA-labelled proteins. Thus, the micro-fibres are developmentally modulated by enzymes of the ECM in embryonic kidney cortex. These findings illustrate the importance of micro-fibres in directing CD ampulla growth.     

水飞蓟宾抑制TNF-α诱导的MMP-9在胃癌细胞中的表达

目的确定在胃癌细胞株中水飞蓟宾对TNF-α诱导的MMP-9表达的影响。方法应用细胞增殖分析、化学抑制剂处理、免疫印迹、流式细胞分析、腺病毒转移等技术完成实验。结果在SNU216和SNU668胃癌细胞中,MMP-9mRNA和蛋白的表达都被TNF-α剂量依赖性地提高。另-方面,TNF—α诱导的MMP-9表达被水飞蓟宾剂量依赖性地抑制。结论在胃癌细胞中水飞蓟宾可减少TNF-α诱导的MMP-9表达。     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.     

Expression of matrix metalloproteinase-9 (gelatinase B) in gouty arthritis and stimulation of MMP-9 by urate crystals in macrophages

To investigate the relevance of gelatinase-B (matrix metalloproteinase 9, MMP-9) in gouty arthritis (GA), we tested the occurrence of MMP-9 in GA patients and cell culture system. Gelatinolytic activity in the synovial fluid (SF) of patients with different kinds of arthritis was assessed by gelatin zymography. A predominant 92-kDa MMP-9 gelatinolytic activity was evident in rheumatoid arthritis (RA) and GA samples, but no activity was observed in osteoarthritis (OA) samples. Among the 53 SF samples (9 RA, 24 GA, and 20 OA) analyzed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP-1) antigen levels by ELISA, MMP-9 antigen levels were elevated tenfold in GA SF compared with OA SF. In addition, GA synovial tissue extracts revealed elevated levels of MMP-9 expression as compared to OA tissue extracts by Western blot and RT-PCR analysis. Immunohistochemical studies demonstrated that MMP-9 immunoreactivity was more intense in GA than in OA synovial tissues. Furthermore, macrophages activation by gouty crystals in vitro was examined. Crystals stimulated MMP-9 gene expression in macrophage cell line and such stimulation was suppressed by PD98059. These findings suggest that the abnormal production of MMP-9 by macrophages is a reflection of the pathological conditions in joints of patients with GA, and that the activation of MMP-9 in the joint is known to play an important role in joint disease.     

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.     

Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.