Unopposed matrix metalloproteinase-9 expression in human tuberculous granuloma and the role of TNF-alpha-dependent monocyte networks

Tuberculosis is characterized by granuloma formation and caseous necrosis, but the factors causing tissue destruction are poorly understood. Matrix metalloproteinase (MMP)-9 (92-kDa gelatinase) secretion from monocytes is stimulated by Mycobacterium tuberculosis (M. tb) and associated with local tissue injury in tuberculosis patients. We demonstrate strong immunohistochemical MMP-9 staining in monocytic cells at the center of granuloma and adjacent to caseous necrosis in M. tb-infected patient lymph nodes. Minimal tissue inhibitor of MMPs-1 staining indicated that MMP-9 activity is unopposed. Because granulomas characteristically contain few mycobacteria, we investigated whether monocyte-monocyte cytokine networks amplify MMP-9 secretion. Conditioned medium from M. tb-infected primary human monocytes or THP-1 cells (CoMTB) stimulated MMP-9 gene expression and a >10-fold increase in MMP-9 secretion by monocytes at 3-4 days (p < 0.009, vs controls). Although CoMTB stimulated dose-dependent MMP-9 secretion, MMP-1 (52-kDa collagenase) was not induced. Anti-TNF-alpha Ab but not IL-1R antagonist pretreatment decreased CoMTB-induced MMP-9 secretion by 50% (p = 0.0001). Anti-TNF-alpha Ab also inhibited MMP-9 secretion from monocytic cells by 50%, 24 h after direct M. tb infection (p = 0.0002). Conversely, TNF-alpha directly stimulated dose-dependent MMP-9 secretion. Pertussis toxin inhibited CoMTB-induced MMP-9 secretion and enhanced the inhibitory effect of anti-TNF-alpha Ab (p = 0.05). Although chemokines bind to G protein-linked receptors, CXCL8, CXCL10, CCL2, and CCL5 did not stimulate monocyte MMP-9 secretion. However, the response to cholera toxin confirmed that G protein signaling pathways were intact. In summary, MMP-9 within tuberculous granuloma is associated with tissue destruction, and TNF-alpha, critical for antimycobacterial granuloma formation, is a key autocrine and paracrine regulator of MMP-9 secretion.     

Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis.

In tuberculosis, matrix metalloproteinase (MMP) secretion is involved in leukocyte migration to sites of infection but in excess may contribute to tissue destruction. We demonstrate that human monocytic THP-1 cells and primary monocytes secrete MMP-1 (52 kD collagenase) when phagocytosing live, virulent M. tuberculosis but not inert latex. The magnitude of MMP-1 secretion was approximately 10-fold less when compared to MMP-9 (92 kD gelatinase) secretion. MMP-1 secretion was also relatively delayed (detected at 24 h vs. 4 h). M. tuberculosis, zymosan or latex stimulate similar TIMP-1 secretion within 8 h and increasing over 24 h. MMP-1/9 secretion was decreased by inhibitors of protein kinase (PK) C, PKA or tyrosine kinases (PTK) in a concentration-dependent manner. In contrast, TIMP-1 secretion was not affected by PKC or PTK blockade and only somewhat reduced by high level PKA inhibition. In summary, M. tuberculosis-infected monocytes secrete MMP-1 at lower concentrations than MMP-9 and such MMP secretion is regulated by multiple upstream signalling pathways which do not control TIMP-1 secretion. Divergent effects of i on MMP and TIMP secretion from monocytes may be important in influencing matrix degradation in vivo.     

Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis

CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung.     

Electronegative LDL induces MMP-9 and TIMP-1 release in monocytes through CD14 activation: Inhibitory effect of glycosaminoglycan sulodexide

Objective

Electronegative LDL (LDL(?)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(?) has never been investigated. The aim of this study was to examine the ability of LDL(?) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion.

Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.     

Interferon-Gamma Increases the Ratio of Matrix Metalloproteinase-9/Tissue Inhibitor of Metalloproteinase-1 in Peripheral Monocytes from Patients with Coronary Artery Disease

Acute coronary syndromes (ACS) may be triggered by acute infections. Systemic production of interferon gamma (IFN-γ) is induced during infection and regulates the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), both important in plaque stability. This study evaluates the effect of IFN-γ on the MMPs/TIMP-1 ratio in cultured monocytes from 30 patients with stable coronary artery disease (CAD), 30 with unstable angina (UA) or non-ST-segment elevation myocardial infarction (NSTEMI), and 30 healthy blood donors. Supernatant concentrations of MMP-1, -2, -9, and TIMP-1 were measured by enzyme-linked immunoassays. Basal concentration of MMP-1 and TIMP-1 was similar between groups, while MMP-2 was higher in healthy individuals and MMP-9 in patients with UA/NSTEMI. Upon IFN-γ stimulation, MMP-9 secretion increased in all groups, while TIMP-1 decreased only in patients with CAD, which in turn result in a strikingly elevation in their mean MMP-9/TIMP-1 ratio. MMP-1/TIMP-1 and MMP-2/TIMP-1 ratios were <1.0 in basal conditions and after stimulation in all groups. Our results suggest that nonstimulated monocytes from patients with stable CAD show a similar behavior than those from healthy individuals. However, stimulation with IFN-γ induces an increase on the MMP-9/TIMP-1 ratio as high as that found in patients with ACS. Thus, it may bring biological plausibility to the association between acute infections and the development of ACS.     

Mycobacterium tuberculosis up-regulates matrix metalloproteinase-1 secretion from human airway epithelial cells via a p38 MAPK switch

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung's tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-alpha and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-alpha synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.     

T lymphocytes activated by persistent viral infection differentially modify the expression of metalloproteinases and their endogenous inhibitors, TIMPs, in human astrocytes: relevance to HTLV-I-induced neurological disease

Activation of T lymphocytes by human pathogens is a key step in the development of immune-mediated neurologic diseases. Because of their ability to invade the CNS and their increased secretion of proinflammatory cytokines, activated CD4+ T cells are thought to play a crucial role in pathogenesis. In the present study, we examined the expression of inflammatory mediators the cytokine-induced metalloproteinases (MMP-2, -3, and -9) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMP-1, -2, and -3), in human astrocytes in response to activated T cells. We used a model system of CD4+ T lymphocytes activated by persistent viral infection (human T lymphotropic virus, HTLV-I) in transient contact with human astrocytes. Interaction with T cells resulted in increased production of MMP-3 and active MMP-9 in astrocytes despite increased expression of endogenous inhibitors, TIMP-1 and TIMP-3. These data suggest perturbation of the MMP/TIMP balance. These changes in MMP and TIMP expression were mediated, in part, by soluble factors (presumably cytokines) secreted by activated T cells. Integrin-mediated cell adhesion is also involved in the change in MMP level, since blockade of integrin subunits (alpha1, alpha3, alpha5, and beta1) on T cells resulted in less astrocytic MMP-9-induced expression. Interestingly, in CNS tissues from neurological HTLV-I-infected patients, MMP-9 was detected in neural cells within the perivascular space, which is infiltrated by mononuclear cells. Altogether, these data emphasize the importance of the MMP-TIMP axis in the complex interaction between the CNS and invading immune cells in the context of virally mediated T cell activation.     

Matrix metalloproteinase expression by endothelial cells in collagen lattices changes during co-culture with fibroblasts and upon induction of decorin expression

EA.hy 926 cells, a derivative of human umbilical vein endothelial cells, in the presence of fibroblasts show the phenomena of angiogenesis, express the proteoglycan decorin and escape apoptosis, when they are maintained in collagen lattices, while fibroblast-free cultures do not show these changes. Virus-mediated decorin expression can substitute for the presence of fibroblasts. Since the expression of matrix metalloproteinases (MMPs) is an essential step in the formation of capillaries, several MMPs and tissue inhibitors of metalloproteinases (TIMPs) were investigated. MMP-1, MMP-2, MMP-9, and the cell-associated MMP-14 were augmented on the protein level in the presence of fibroblasts. No effect was seen with respect to MMP-3, TIMP-1, and TIMP-2. Semiquantitative RT-PCRs of endothelial cells in co-culture revealed a 7-, 19-, and 11-fold increase for mRNAs of MMP-1, MMP-2, and MMP-14 after six days, respectively. Virus-mediated decorin expression also was accompanied by an up-regulation of these MMPs. The expression of MMP-1 mRNAs increased 5-fold after 2 days and gradually declined thereafter. In contrast, MMP-2 and MMP-14 showed a 7-fold and a 14-fold increase on day two which returned to basal levels within 24 h, indicating that the expression of MMP-1 is differentially regulated from MMP-2 and MMP-14. In spite of the upregulation of the proteases, an enhanced degradation of decorin was not observed. These results indicate that the expression of decorin is a sufficient signal in EA.hy 926 cells for a finely tuned induction of selected MMPs which are involved in angiogenesis whereas the up-regulation of MMPs does not lead to the degradation of the responsible proteoglycan.     

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodelling in the brain and a continuous requirement for peripheral leucocyte migration within the CNS (central nervous system). In the present study, we investigate the role of MMPs (matrix metalloproteinases) and their inhibitors in T-cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases-1), was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T-cells. In addition, infiltrating T-cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1-deficient mice had a decrease in perivascular accumulation of lymphocyte populations, yet an increase in the proportion of CD4+ T-cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Taken together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.     

A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

Background

Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons.

Principal Findings

We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, βIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1.

Conclusions

Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.     

Leukocyte matrix metalloproteinase-9 is elevated and contributes to lymphocyte activation in type I diabetes

Degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs) is integral to cell migration and tissue remodeling in diabetes mellitus and its complications. MMPs also regulate the function of leukocytes via proteolytic processing of cytokines/chemokines. In this study, we measured the production of MMP-9 and its natural tissue inhibitor (TIMP)-1 by leukocytes isolated from human type I diabetic patients. MMP-9 was also detected in serum and splenocytes from non-obese diabetic (NOD) and BALB/c mice. MMP-9 was markedly elevated in leukocytes from diabetics compared to non-diabetic controls. TIMP-1 production was also enhanced in leukocytes from diabetics, but substantially less than MMP-9, with the MMP-9/TIMP-1 ratio being 1.6-fold higher in neutrophils and 3-fold higher in monocytes than controls. Interleukin (IL)-2 or lipopolysaccharide (LPS) treatment increased MMP-9 production in leukocytes from both diabetics and normal controls, whereas insulin decreased MMP-9 expression. Recombinant MMP-9 stimulated the proliferation of mouse splenocytes from NOD or BALB/c and a MMP-9 inhibitor dose-dependently inhibited splenocyte proliferation. In conclusion, our results demonstrate firstly that MMP-9 expression is elevated in leukocytes from type I diabetic patients and NOD mice and secondly, that MMP-9 elevates proliferation of mouse splenocytes. These data suggest that elevated leukocyte MMP-9 may contribute to the pathogenesis of type I diabetes and its associated complications.     

Chemokine-Induced secretion of gelatinase B in primary human monocytes.

Chemokines help control normal leukocyte trafficking as well as their infiltration into tissues during acute and chronic inflammation. Matrix metalloproteinases (MMPs) help support the extravasation and infiltration of leukocytes through limited proteolysis of basement membranes and matrix material. The effect of the chemokines RANTES/CCL5, MCP-1/CCL and SDF-1/CXCL12 on secretion of the matrix metalloproteinase B and its endogenous inhibitor TIMP-1 was studied. RANTES/CCL5 and SDF-1/CXCL12 were found to induce MMP-9 secretion in primary human monocytes while TIMP-1 secretion was not affected. RANTES/CCL5 effects were mediated through CCR1 because the CCR1 antagonist BX471 was found to effectively block RANTES/CCL5-induced MMP-9 secretion.     

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.     

Identification of a matrix-degrading phenotype in human tuberculosis in vitro and in vivo

Tuberculous meningitis is characterized by cerebral tissue destruction. Monocytes, pivotal in immune responses to Mycobacterium tuberculosis, secrete matrix metalloproteinase-9 (MMP-9), which facilitates leukocyte migration across the blood-brain barrier, but may cause cerebral injury. In vitro, human monocytic (THP-1) cells infected by live, virulent M. tuberculosis secreted MMP-9 in a dose-dependent manner. At 24 h, MMP-9 concentrations increased 10-fold to 239 +/- 75 ng/ml (p = 0.001 vs controls). MMP-9 mRNA became detectable at 24--48 h. In contrast, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) gene expression and secretion were similar to constitutive levels from controls at 24 h and increased just 5-fold by 48 h. In vivo investigation revealed MMP-9 concentration per leukocyte in cerebrospinal fluid (CSF) from tuberculous meningitis patients (n = 23; median (range), 3.19 (0.19--31.00) ng/ml/cell) to be higher than that in bacterial (n = 12; 0.23 (0.01--18.37) ng/ml/cell) or viral meningitis (n = 20; 0.20 (0.04--31.00) ng/ml/cell; p < 0.01). TIMP-1, which was constitutively secreted into CSF, was not elevated in tuberculous compared with bacterial meningitis or controls. Thus, a phenotype in which MMP-9 activity is relatively unrestricted by TIMP-1 developed both in vitro and in vivo. This is functionally significant, since MMP-9 concentrations per CSF leukocyte (but not TIMP-1 concentrations) were elevated in fatal tuberculous meningitis and in patients with signs of cerebral tissue damage (unconsciousness, confusion, or neurological deficit; p < 0.05). However, MMP-9 activity was unrelated to the severity of systemic illness. In summary, M. tuberculosis-infected monocytic cells develop a matrix-degrading phenotype, which was observed in vivo and relates to clinical signs reflecting cerebral injury in tuberculous meningitis.     

Differential regulation of gelatinase A and B and TIMP-1 and -2 by TNFalpha and HIV virions in astrocytes

Changes in the fine balance between matrix metalloproteinases and their tissue inhibitors, which drives extracellular matrix turnover, may be critical to central nervous system inflammation in HIV infection as well as in neurotoxicity. Although they do not produce virus when infected by HIV, astrocytes may be directly affected by the virion, because some viral proteins are known to transduce signaling in brain cells and are also sensitive to the major proinflammatory cytokine TNFalpha. We therefore studied the effects of HIV and TNFalpha on MMP-2, MMP-9 and their inhibitors, TIMP-1 and TIMP-2, in astrocytes, by zymography and ELISA, respectively, or by RT-PCR for both of them. HIV slightly increased the production of pro-MMP-2 and pro-MMP-9 by astrocytes, in a dose-dependent manner. TNFalpha strongly induced pro-MMP-9. TIMP-1 and TIMP-2 levels were affected only slightly, if at all, by HIV and TNFalpha. Thus, astrocyte/HIV contact may lead to extracellular matrix activation, which may be strongly amplified by the inflammatory response. Our data strongly suggest that, besides their physiological production of MMP-2, astrocytes would be a major source of MMP-9 in the inflamed brain.     

C-type natriuretic peptide suppresses mesangial proliferation and matrix expression via a MMPs/TIMPs-independent pathway in vitro

C-type natriuretic peptide (CNP) acts mainly in a local, paracrine fashion to regulate vascular tone and cell proliferation. Although several in vivo studies have demonstrated that CNP exerts an inhibitory effect on mesangial matrix generation, a limited number of reports exist about the anti-extracellular matrix (ECM) accumulation effect of CNP and its underlying mechanisms in mesangial cells (MCs) in vitro. In this study, human MCs were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100?pM) for 24, 48 and 72?h, respectively. CNP administration significantly suppresses MCs proliferation and collagen (Col)-IV expression in a time- and dose-dependent manner. In addition, the study presented herein was designed as a first demonstration of the regulative effects of CNP on the metabolisms of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in MCs in vitro, and found that: (1) CNP administration significantly decreased the secretion and expression of MMP-2 and MMP-9 in the cultured MCs; (2) the secretion and expression of TIMP-1 progressively elevated after treatment with CNP for 24 and 48?h, whereas declined at later time point; (3) CNP expression was negatively correlated with MMP-2 and MMP-9 expression; (4) the balance of MMPs/TIMPs was shifted toward the reduction in MMP-2 and MMP-9 activity and/or the increment in TIMP-1 expression, which could not account for the down-regulation of Col-IV expression in CNP-treated MCs. In conclusion, CNP suppresses mesangial proliferation and ECM expression via a MMPs/TIMPs-independent pathway in vitro.