Tissue Inhibitor Of Metalloproteinase-1 Production Research Articles

Mammary carcinoma cells over-expressing tissue inhibitor of metalloproteinases-1 show enhanced vascular endothelial growth factor expression.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) has at least 2 independent functions, i.e., regulation of matrix metalloproteinases and erythroid-potentiating activity. We investigated the effects of TIMP-1 over-expression on tumor growth, using cloned lines derived from a TIMP-1-transfected rat breast carcinoma cell line. The in vitro growth rate of the TIMP-1-transfected clones was indistinguishable from that of the control. In contrast, the highest TIMP-1-producing clone (159.0 ng/ml), designated as T-H, formed 4.6-fold larger s.c. tumors than did the control after 14 days. Tumors derived from an intermediate TIMP-1-producing clone (45.4 ng/ml), designated as T-M, were 1.9-fold larger than the control. TIMP-1 over-expression was associated with increased vascular endothelial growth factor (VEGF) expression, vascularization and proliferative activity of the s.c. tumors. Similar to the rat breast carcinoma cells, transfection of TIMP-1 cDNA into the human breast carcinoma cell line MCF-7 resulted in up-regulation of VEGF, with a linear relationship between TIMP-1 and VEGF production in 9 cell clones examined. There was, however, no change in VEGF expression when the rat and human breast carcinoma cell lines were exposed to exogenous recombinant TIMP-1. Our findings suggest that over-expression of TIMP-1 confers growth advantage on breast carcinoma cells in vivo and that up-regulation of VEGF expression may play an important role in this TIMP-1-mediated, growth-stimulating effect.

Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase by myofibroblasts: Morphological study on corneal wound healing

The matrix metalloproteinases (MMPs) and the tissue inhibitor of metalloproteinases (TIMPs) regulate the extracellular matrix and are important in the process of connective tissue remodeling. In this study, we investigated the expression of MMPs and TIMP-2 by myofibroblasts (MF) originating from keratocytes during the healing of alkali-burned and lacerated rabbit corneas. In light and/or electron microscopic immunohistochemistry, the MMP-1, 2, 9 and TIMP-2 antibodies reacted with MF in both types of corneal wounds. The clear expression of MMP-1 and the relatively faint immunoreaction of MMP-2 were observed in both types of healing from one week after wounding, while MMP-9 appeared from 3 weeks after injury. In addition, the levels of MMP-2 and 9 were increased from 4 weeks after alkali-burned injury. The clear expression of TIMP-2 was observed from 1 week after both types of wounds. These findings indicate that MF is deeply involved in degrading some of the key matrix proteins (such as type I collagen), and may play an important role in tissue remodeling in corneal wounds through production of MMPs and TIMP.

Immunolocalization of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in radicular cysts.

To investigate the mechanisms involved in expansion of radicular cysts, monoclonal antibodies against interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were used to localize the sites of MMP-1 and TIMP-1 expression in 30 radicular cysts. Positive MMP-1 staining was detected in the lining epithelium and subepithelial fibroblasts, macrophages, endothelial cells and osteoblasts/osteocytes in all specimens. Positive TIMP-1 staining was identified in osteoblasts/osteocytes and endothelial cells of all specimens, and in the lining epithelium and subepithelial fibrous connective tissue wall of five radicular cysts with an intense inflammatory cell infiltrate. The number and distribution of positive cells for MMP-1 or TIMP-1 varied widely among individual specimens, but strong immunostaining was constantly detected at sites with prominent subepithelial inflammation. Results here support the hypothesis that MMP-1 may play an important role in the expansion of radicular cysts. The absence of TIMP-1 expression in lining epithelium and subepithelial fibroblasts and macrophages in most cases studied indicated that an imbalance between MMP-1 and TIMP-1 production may lead to radicular cyst expansion.

Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc.

This study is a unique in vitro study on the effects of hydrostatic pressure on human intervertebral disc metabolism. To investigate the effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc. Mechanical stress and hydrostatic pressures influence proteoglycan and protein synthesis rates in bovine articular cartilage and coccygeal discs. However, the mechanism of matrix synthesis regulation of the intervertebral disc under mechanical stress has not been elucidated. Twenty-eight human lumbar intervertebral discs obtained from surgery and from cadavers at autopsy were used. Each tissue fraction was charged with medium in a plastic syringe and placed in a water-filled hydrostatic pressure-control vessel. The hydrostatic pressures applied were 1 (control), 3, and 30 atm (atm = atmospheres) for 2 hours. The proteoglycan and protein synthesis rates were determined by radioisotope incorporation. The production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were measured by a one-step enzyme immunoassay method using monoclonal antibodies. Three atm pressure stimulated proteoglycan synthesis rates in the nucleus pulposus and inner anulus (n = 14 in each tissue). Compared with the control group, 30 atm pressure significantly inhibited proteoglycan synthesis in the inner anulus (P = 0.011). In the nucleus pulposus, matrix metalloproteinase-3 production was stimulated at a pressure of 30 atm relative to 3 atm (P = 0.014, n = 16 in each tissue). The highest tissue inhibitor of metalloproteinases-1 production showed highest values at 3 atm pressure in the inner anulus (n = 16 in each tissue). The results suggest that hydrostatic pressure influences intervertebral disc cell metabolism. A physiologic level of hydrostatic pressure (3 atm) may act as an anabolic factor for stimulation of proteoglycan synthesis and tissue inhibitor of metalloproteinases-1 production. This may be essential for maintaining the matrix of the disc. If the pressure was 30 atm or more or 1 atm or less, a catabolic effect will be predominant, with reduction of proteoglycan synthesis rate and increase of matrix metalloproteinase-3 production. Abnormal hydrostatic pressure, therefore, may accelerate disc degeneration.

Effect of transforming growth factor-β on the plasminogen activator system in cultured first trimester human cytotrophoblasts

Plasminogen activators (PAs) play an important role in facilitating trophoblast invasion of the uterus and in the maintenance of blood fluidity within the placental intervillous spaces. We previously found that transforming growth factor-β (TGF-β), produced mainly by the decidua, inhibits first trimester trophoblast invasiveness at least partly via induction of tissue inhibitor of metalloproteinases-1 production and secretion by the trophoblasts. The present study examined whether TGF- β 1 affects PA and plasminogen activator inhibitor-1 (PAI-1 ) production by cultured human cytotrophoblasts. Immortalized first trimester human cytotrophoblasts (HTR-8/SVneo) were cultured in the absence or presence of TGF- β 1 (0–10 ng/ml) for 24 h, after which the levels of urokinase-type PA (uPA), PAI-1 and uPA activity in the serum-free conditioned media were determined by enzyme-linked immunosorbent assay (ELISA), protein zymography, and a chromogenic uPA activity assay. Cellular PAI-1 mRNA levels were also determined in cultures following a 24-h incubation with a single dose (5 ng/ml) of TGF- β 1. Presence of TGF- β 1 at 1 ng/ml resulted in a greater than 12-fold reduction in the levels of total uPA as determined by ELISA. Furthermore, released uPA activity levels in similar cultures incubated with 5 ng/ml of TGF- β 1 were reduced to 35 per cent of control values. In contrast, cultures incubated with as little as 0.1 ng/ml TGF-β exhibited a 63 per cent increase in the levels of secreted PAI-1 protein. Similarly, both the 2.2- and 3.0-kb PAI-1 cellular mRNA species were elevated in trophoblast cells incubated with 5 ng/ml TGF- β 1 when compared with control cells. Thus, it appears that the reduced uPA activity observed in the cultures incubated with TGF- β 1 is due to reduced secretion of uPA and increased PAI-1 production and secretion. These results suggest that TGF-β may also exert its anti-invasive effect by down-regulating trophoblast-derived PA activity. Through its effects on the PA system, TGF-β may also play an indirect role in the regulation of uteroplacental blood flow.

Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) May Be an Autocrine Growth Factor in Scleroderma Fibroblasts.

In scleroderma (systemic sclerosis, SSc), an autoimmune disorder in which excessive extracellular matrix is deposited in skin and internal organs, one of the suggested contributory factors to the development of fibrosis is a decrease in collagenase activity that may be related to levels of serum tissue inhibitors of metalloproteinases 1 (TIMP-1). We recently reported that the serum TIMP-1 levels in SSc patients were elevated compared with normal controls. To determine the biologic significance of TIMP-1 in SSc, we compared the proliferative effects of TIMP-1 between normal and SSc fibroblasts. TIMP-1 showed significant mitogenic activity for both normal and SSc fibroblasts. The mitogenic responses to TIMP-1 (33-100 ng/ml) in SSc fibroblasts, however, were significantly greater than those in normal controls and were completely neutralized in the presence of anti-TIMP-1 IgG. Moreover, anti-TIMP-1 IgG partially but significantly blocked the basal mitogenic activities of SSc fibroblasts. SSc fibroblasts produced increased amounts of TIMP-1 relative to normal fibroblasts, as confirmed by western blotting, ELISA, and RT-PCR techniques. In contrast, transforming growth factor beta1 (TGF-beta1) upregulated TIMP-1 production in normal fibroblasts but not in SSc fibroblasts with elevated spontaneous secretion of TIMP-1. These observations suggest that TIMP-1 may play an important role as an autocrine growth factor in the fibrotic process in SSc.

Matrixmetalloproteinases and tissue inhibitor of metalloproteinases production in bovine placenta and uterine tissues during early pregnancy

Matrixmetalloproteinases and tissue inhibitor of metalloproteinases production in bovine placenta and uterine tissues during early pregnancy

Partial purification and amino acid sequence analysis of endometriosis protein-II (ENDO-II) reveals homology with tissue inhibitor of metalloproteinases-1 (TIMP-1).

De novo synthesized endometriosis protein-II (ENDO-II; M(r) 28,000 to 32,000; pI 7.0 to 9.0) was partially purified from rat endometriotic tissue culture media using affinity chromatography and two-dimensional SDS-PAGE. The protein was electrophoretically transferred to polyvinyl difluoride membranes which were stained with Coomassie blue R-250. The stained protein corresponding to ENDO-II (M(r) 28,000 to 32,000; pI 7.0 to 9.0) was cut from the membranes for amino acid sequencing. Partial amino acid sequence was determined by automated Edman degradation using a gas phase sequencer and phenylthiohydantoin analyzer. Sequence analysis of ENDO-II yielded 25 residues: C S C A P T H P Q T A F C N S D L V I R K F M G. Comparison to sequence databanks demonstrated significant homology with rat (100%) and human (84%) tissue inhibitor of metalloproteinases-1 (TIMP-1). Western blot analysis using a TIMP-1 antibody confirmed amino acid sequence analysis. In conclusion, ENDO-II shares sequence homology with TIMP-1 and cross-reactivity with TIMP-1 antibody and subsequently identifies production of TIMP-1 by endometriotic tissues. The synthesis and secretion of TIMP-1 by endometriosis may derange the normal proteolytic milieu of the peritoneal cavity and contribute to the etiology and underlying physiological sequelae associated with the disease endometriosis.

IL-10 inhibits metalloproteinase and stimulates TIMP-1 production in human mononuclear phagocytes.

Human mononuclear phagocytes can modulate the turnover of extracellular matrix by producing metalloproteinases such as 92-kD gelatinase and interstitial collagenase as well as the tissue inhibitor of metalloproteinases (TIMP). We have previously reported that IL-4 and IFN gamma released by lymphocytes suppress metalloproteinase biosynthesis in macrophages without affecting TIMP production (Lacraz, S., L. Nicod, B. C. de Rochementeix, C. Baumberger, J. Dayer, and H. Welgus. 1992. J. Clin. Invest. 90:382-388.; Shapiro, S. D., E. J. Campbell, D. K. Kobayashi, and H. G. Welgus 1990. J. Clin. Invest. 86:1204-1210). Like IL-4, IL-10 is secreted by Th2 lymphocytes and is inhibitory to several macrophage functions. In the present study, IL-10 was tested and compared to IL-2, IL-4, IL-6, and IFN gamma for its capacity to modulate synthesis of 92-kD gelatinase, interstitial collagenase and TIMP in human macrophages and monocytes. We found that IL-10, just like IL-4, inhibited the production of 92-kD gelatinase and blocked LPS-, as well as killed Staphylococcus aureus-induced, interstitial collagenase production. The principal finding of this study, however, was that IL-10, in distinction to IL-4, produced a dose-dependent stimulation in the biosynthesis of TIMP-1. TIMP-2 production was not affected. IL-10 regulated the expression of 92-kD gelatinase and TIMP-1 at the pretranslational level. Furthermore, IL-10 regulation was cell type-specific, as it had no effect on the production of metalloproteinases or TIMP by human fibroblasts. In summary, IL-10 has a potent and unique effect upon tissue macrophages and blood monocytes by enhancing TIMP-1 production while decreasing metalloproteinase biosynthesis.

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells.

The hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.

Agonist-Induced Expression of Tissue Inhibitor of Metalloproteinases and Metalloproteinases by Human Macrophages Is Regulated by Endogenous Prostaglandin E2 Synthesis

The regulatory effect of endogenously synthesized eicosanoid metabolites on the expression of tissue inhibitor of metalloproteinases (TIMP), interstitial collagenase, and 92-kDa gelatinase by human macrophages was examined. TIMP and metalloproteinase production were stimulated with three agonists that produce distinct patterns of eicosanoid synthesis: lipopolysaccharide (10 micrograms/ml), denatured collagen (10 micrograms/ml), or zymosan (1 mg/ml). Indomethacin (3 micrograms/ml) or MK886 (3 microM), a specific inhibitor of 5-lipoxygenase, was used to examine the role of endogenous metabolites of arachidonic acid. Regardless of the agonist used, TIMP production by macrophages was inhibited 65% by indomethacin, synthesis of interstitial collagenase was reduced 70%, and expression of 92-kDa gelatinase was decreased 40%. In contrast, inhibition of leukotriene synthesis had no effect on metalloproteinase or TIMP production. The agonist-stimulated increase in TIMP and collagenase production was directly correlated to the cumulative prostaglandin E2 level induced by the agonist used. However, if response to an agonist was poor, the exogenous addition of prostaglandin E2 could not increase TIMP or collagenase production more than twofold, indicating an important permissive effect of the agonist on the regulation of each protein's expression. The mechanism of indomethacin inhibition of TIMP and collagenase production was studied by labeling the cells with [35S]-methionine and performing immunoprecipitation using specific antiserum. Indomethacin markedly inhibited the lipopolysaccharide-induced biosynthesis of both TIMP and collagenase. Northern analysis revealed parallel suppression of TIMP and collagenase steady-state mRNA levels by indomethacin, indicating pretranslational control. The regulation of inflammatory-cell TIMP and interstitial collagenase expression by prostaglandin E2 suggests that therapy inhibiting the cellular response to prostaglandins may be useful in cutaneous and systemic disease states involving macrophage-mediated connective-tissue destruction.

Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors.

The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.

Effect of a Benzylidene Derivative, Novel Antirheumatic Agent, on the Production of Tissue Inhibitor of Metalloproteinases.

We investigated the effects of PAL (protocatechualdehyde), a metabolite of ACP (3,4-diacetoxy benzylidene diacetate) which is a candidate as a novel antirheumatic agent, on the production of a tissue inhibitor of metalloproteinases (TIMP) using a primary chondrocyte culture from bovine nasal septum cartilage. The addition of human recombinant interleukin-1 alpha (hrIL-1 alpha) induced proteoglycan (PG) depletion from a chondrocyte matrix. PAL significantly reduced the induction of PG depletion. HrIL-1 alpha increased the casein-degrading activity, matrix metalloproteinase (MMP) activity, and the level of TIMP secretion in the culture media. PAL significantly reduced the casein-degrading activity and further increased TIMP secretion under hrIL-1 alpha stimulation. Phorbol myristate acetate (PMA) also increased the level of TIMP secretion, and staurosporine, a specific inhibitor of protein kinase C (PKC), reduced the TIMP secretion to the control level under hrIL-1 alpha or PMA stimulation. Furthermore, PAL showed a significant increase following treatment with a low concentration of PMA which alone did not increase TIMP production. These findings suggest that the activation of the PKC pathway plays an important role in TIMP production and that PAL increases the level of TIMP production through an additive or synergistic effect with the activation of the PKC pathway. In conclusion, these findings demonstrate that the inhibition of the MMP activity and the increase of TIMP production by PAL contribute to the inhibition of PG depletion in a primary chondrocyte culture from bovine nasal septum cartilage.

Molecular mechanisms regulating the production of collagenase and TIMP in U937 cells: Evidence for involvement of delayed transcriptional activation and enhanced mRNA stability

We have used the human promonocyte-like U937 cell line as a model to study the regulation of interstitial collagenase and tissue inhibitor of metalloproteinases (TIMP) during mononuclear phagocyte development. Our results show that differentiation of U937 cells with exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) induces a temporally delayed (16-24 h) but marked increase in the biosynthesis and secretion of interstitial collagenase and TIMP. Similarly, steady-state mRNA levels for both proteins rose dramatically during the period of exposure, but again after considerable time delay (12-16 h). For interstitial collagenase, induction was transcriptionally regulated as demonstrated by nuclear run-on experiments, and required the synthesis of proteins as indicated by cycloheximide treatment. However, transcriptional activation of collagenase was never observed prior to 10-12 h; since c-fos is rapidly induced in U937 cells and largely disappears by 2 h (Mitchell et al., 1985), our data strongly suggest that collagenase induction in this system cannot be explained simply or entirely by an AP-1-dependent mechanism. Although TIMP steady-state mRNA levels also increased substantially with cellular differentiation, no transcription was detected by run-on experiments. However, TPA exposure markedly prolonged the half-life of TIMP mRNA from 4 h to > 20 h. While cycloheximide treatment completely blocked TPA-mediated induction of collagenase mRNA, it only marginally interfered with simultaneously induced TIMP mRNA levels. Our results demonstrate that differentiation of U937 monocytic cells is accompanied by markedly enhanced production of both interstitial collagenase and TIMP. However, there are multiple, and perhaps differing, molecular mechanisms regulating these responses.

Interleukin-6 does not regulate interstitial collagenase, stromelysin and tissue inhibitor of metalloproteinases synthesis by cultured human fibroblasts

We have examined the effect of interleukin-6 (IL-6) on the synthesis of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) by human skin fibroblasts, both at protein and messenger RNA levels. IL-6 did not modulate the production of MMPs and TIMP. Furthermore IL-6 did not modify the stimulatory effect exerted by interleukin-1 (IL-1) on the expression of MMPs and TIMP. These results strongly suggest that IL-6 is not involved in the extracellular matrix breakdown, either directly by acting on cell production of MMPs or TIMP, or indirectly by modulating the effect of IL-1 on the synthesis of MMPs and TIMP.

Production of Collagenase and Tissue inhibitor of Metalloproteinases by Fibroblasts Derived from Normal and Fibrotic Human Lungs

Several experiments have demonstrated low collagenolytic activity during the development of pulmonary fibrosis. In order to determine if fibroblasts play a role in this alteration, procollagenase and tissue inhibitor of metalloproteinases (TIMP) were quantified in fibroblasts derived from 12 human lung specimens (normal = 6, idiopathic pulmonary fibrosis [IPF] = 6). Under basal conditions, three cell strains from normal and three from fibrotic lung specimens did not synthesize collagenase and a similar number of normal and IPF-derived fibroblast strains produced the enzyme. However, the rate of enzyme synthesis among normal and fibrotic collagenase producing fibroblasts exhibited significant differences. Thus, whereas normal fibroblasts produced more than 300 ng/ml, fibrotic lung fibroblasts secreted approximately half of this amount (115 +/- 67 ng/ml). Phorbol myristate acetate (PMA) enhanced collagenase production in all of the 12 lung fibroblast lines tested. In four IPF fibroblasts, PMA increased collagenase secretion close to those of normal stimulated lung fibroblasts; however, a lower induction was observed in cell strains from two fibrotic lung specimens. There was a wide variation in TIMP production both in normal and fibrotic lung fibroblasts, and no statistically significant difference was observed. Under basal conditions, TIMP levels ranged from 329 to 16,911 ng/ml in normal lung cells, and from 377 to 17,557 in fibrotic lung fibroblasts. PMA induced a severalfold increase in all cell lines. These results suggest that there are subpopulations of lung fibroblasts with different potential to produce collagenase and TIMP in vitro, and that the predominance of low collagenase-producing subsets may contribute to the development of fibrosis.

Ultraviolet A Irradiation Stimulates Collagenase Production in Cultured Human Fibroblasts

This study was designed to investigate the biochemical mechanisms responsible for the connective tissue changes seen in actinically damaged skin, which is characterized histologically by diminution and ultrastructural alterations of collagen fibrils and deposition of elastotic material in the papillary dermis. We hypothesized that ultraviolet light could stimulate synthesis of interstitial collagenase in the skin, resulting in collagen degradation. Monolayer cultures of human fibroblasts or keratinocytes were irradiated with ultraviolet A (UVA) or ultraviolet B (UVB) radiation and interstitial collagenase or its inhibitor, TIMP (tissue inhibitor of metalloproteinases) assessed in the conditioned medium with Western immunoblots 24 h after irradiation. Northern blot analysis of the irradiated fibroblasts with a cDNA probe representing collagenase was also performed. Cell viability was greater than 90% with all doses of UV radiation studied. A dose-related increase in immunoreactive collagenase was detected in the medium of fibroblasts irradiated with 0-10 J/cm2 of UVA radiation as well as a parallel increase in the collagenase mRNA in the irradiated cells. UVA radiation stimulated collagenase synthesis in both neonatal and adult fibroblasts. TIMP production in UVA-irradiated fibroblasts increased to a lesser degree than did collagenase and its increase did not parallel the increase in collagenase. UVB (0-100 mJ/cm2) did not stimulate collagenase production by fibroblasts. In contrast to the stimulation of collagenase production by fibroblasts, a slight decrease in immunoreactive collagenase was seen in UVA-irradiated keratinocytes. These data suggest that direct stimulation of collagenase synthesis by human skin fibroblasts by UVA radiation may contribute to the connective tissue damage induced by ultraviolet radiation leading to photoaging.

Suppression of metalloproteinase biosynthesis in human alveolar macrophages by interleukin-4.

To study the interaction of lymphocytes and macrophages in the control of extracellular matrix turnover, we determined the effects of several soluble T cell products on mononuclear phagocyte production of metalloproteinases. Cytokines including IL-2, IL-4, IL-6, tumor necrosis factor alpha (TNF alpha), GM-CSF, and IFN-gamma were each tested for capacity to modulate macrophage metalloproteinase and tissue inhibitor of metalloproteinases (TIMP) expression. The addition of IL-4 to cells cultured under basal conditions caused a dose-dependent suppression in the release of 92-kD type IV collagenase without affecting TIMP production. 92-kD enzyme secretion was inhibited by 50% with 1-2 ng/ml of IL-4 and by 90% with 10 ng/ml of IL-4. When cells were first exposed to killed Staphylococcus aureus to induce metalloproteinase production, IL-4 potently blocked the stimulated release of both interstitial collagenase and 92-kD type IV collagenase, again without effect upon TIMP. Metabolic labeling experiments and Northern hybridizations demonstrated that IL-4 exerted its action at a pretranslational level. Furthermore, IL-4 possessed the capacity to inhibit metalloproteinase expression even in the relatively immature peripheral blood monocyte. As reported previously (Shapiro, S. D., E. J. Campbell, D. K. Kobayashi, and H. G. Welgus. 1990. J. Clin. Invest. 86:1204), IFN-gamma suppressed constitutive macrophage production of 92-kD type IV collagenase. Despite the frequent antagonism observed between IL-4 and IFN-gamma in other systems, the combination of these two agents lowered metalloproteinase biosynthesis dramatically, whereas IL-4 opposed the IFN-gamma-stimulated production of cytokines (IL-1 and TNF alpha). IL-6 had only minimal effect upon metalloproteinase production, but appeared to specifically augment TIMP release. In summary, cytokines released by activated T cells may profoundly reduce the capacity of the macrophage to mediate extracellular matrix degradation.

Tumor necrosis factor-alpha stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells.

To investigate the role of tumor necrosis factor-alpha (TNF alpha) in advanced collagenolysis and degradation of connective tissue components in preterm parturition, the effects of human recombinant TNF alpha (hrTNF alpha) on the production of matrix metalloproteinase 1 (MMP-1)/tissue collagenase, MMP-3/stromelysin, tissue inhibitor of metalloproteinases (TIMP), urokinase type-plasminogen activator (uPa) and prostaglandin (PG) E2 in human chorionic cells were examined in vitro. Human chorionic cells, but not amniotic cells, were found to respond to macrophage-conditioned medium (contains mainly interleukin 1) to produce MMP-1 and MMP-3. This indicated that the chorionic cell is one of the MMP-producing cells of fetal membranes. When confluent chorionic cells were treated with hrTNF alpha, the production of MMP-1 and MMP-3 as well as of uPa and PGE2 was greatly increased in a dose-dependent manner. In contrast, the production of TIMP was suppressed by hrTNF alpha. These results suggested that TNF alpha may participate in destruction of collagen and other connective tissue matrix components of fetal membranes and in promotion of uterine contractility in preterm parturition with intraamniotic infection.

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10 −7 to 10 −5 M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.