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PubMed1987

Biosynthesis of glycosaminoglycans by trabecular meshwork cells in vitro.

We compared the incorporation of radioactive precursors into glycosaminoglycans by cynomolgus monkey trabecular meshwork cells in tissue culture and in organ culture. Hyaluronic acid and a variety of sulfated glycosaminoglycans were synthesized in both systems. In organ cultures, the ratio of chondroitin 6- to chondroitin 4-sulfate was higher than that found in tissue cultures. Also, a greater proportion of total glycosaminoglycans found in the organ culture medium was represented by hyaluronic acid. The higher production of chondroitin 6-sulfate and hyaluronic acid, as noted in some embryonic systems, suggested that cells in organ cultures may resemble an acute wound healing or an early developmental state more closely than did cells in tissue cultures.

Animals↗
PubMed1996

Natriuretic peptide receptors on human trabecular meshwork cells.

The effects of natriuretic peptides on cGMP formation and [125I]ANP binding in human trabecular meshwork cells were investigated. CNP at 1 microM stimulated cGMP formation approximately 18-25 fold, with a half maximal effective concentration approximately 20-30nM. BNP at 1 microM stimulated approximately 7 fold, while ANP stimulated cGMP formation 2-fold at 1 microM but had little or no effect at concentrations below 1 microM. Displacement binding of [125I]ANP to intact TM cells in the presence of unlabeled ANP indicated a single binding site with a dissociation constant approximately 0.15nM.c-ANP, which binds specifically to natriuretic peptide C receptors, displaced > 95% [125I]ANP binding to surface receptor sites with a half-maximal effective concentration comparable to that of ANP or BNP. c-ANP had no inhibitory effect on CNP stimulation of cGMP formation. The data suggest that human TM cells possess natriuretic peptide B receptors as the primary guanylyl cyclase-containing subtype and C receptors as the numerically predominant subtype of natriuretic peptide receptors.

Adult↗
PubMed1982

Schwalbe line's cells: a new cell type in the trabecular meshwork of Macaca mulatta.

In the eye of Macaca mulatta, at the anterior end of the trabecular meshwork, just beneath the ridge known as Schwalbe line in gonioscopy, cells are present that contain secretory inclusions. These cells have been termed Schwalbe line's cells. They form a discontinuous cord, oriented circumferentially at the corneal periphery, deep to the endothelial lining of the anterior chamber. They are characterized by a prominent Golgi apparatus and by two types of secretory granules: round bodies, up to 0.6 micron in diameter and containing moderately dense material, and larger inclusions, up to 1 micron in diameter and consisting of stacks of osmiophilic lamellae. Membrane whorls and fragments are also commonly found in the spaces between the cells and possibly arise from exocytosis of the lamellar bodies. Schwalbe line's cells have been observed in young as well as in old animals. They are joined to one another by gap junctions and puncta adhaerentia. Because their cytoplasmic inclusions bear a striking morphological resemblance to the multilamellar bodies or cytosomes of the type II alveolar epithelial cells of the lung, the hypothesis is advanced that Schwalbe line's cells produce a phospholipid material that is released in the aqueous humor and thus facilitates its movement through the tissues of the sclerocorneal angle.

Animals↗
PubMed1989

Analysis of the proteins of calf and cow trabecular meshwork: development of a model system to study aging effects and glaucoma.

The proteins from the trabecular meshwork (TM) of calf and cow eyes were analysed to determine if differences in composition were present and to examine whether this tissue could be used as a framework for the study of glaucoma. Differences in polypeptide composition or amount of protein could be detected with extractions using either acetic acid, neutral buffers and urea, or guanidine hydrochloride. In general, the results suggest that an aggregation of proteins may be occurring with aging. The acid-soluble fraction of both calf and cow TM resembled older human TM specimens with the most prominent protein around 68 kD. To test the utility of the bovine TM system, a mixed function oxidation system was used to determine how the proteins of the TM would react to oxidative stress. Aggregation of the proteins in calf TM as well as actin could be demonstrated, consistent with the idea that the aggregation seen in the cow TM might be the result of oxidation of this tissue. The present study lays a foundation for future work on bovine TM and is consistent with the hypothesis that aging changes in this tissue might be a result of oxidative processes.

Actins↗
PubMed2000

Genes upregulated in the human trabecular meshwork in response to elevated intraocular pressure.

PURPOSE: To identify genes upregulated in perfused, intact human trabecular meshwork (TM) in response to elevated intraocular pressure (IOP). METHOD: Two pairs of anterior segments of normal human eyes from postmortem donors were placed in culture and perfused 24 hours at constant flow (3 microl/min). After reaching baseline, the flow of one eye from each pair was raised to obtain an incremental pressure (deltaP) of 50 mm Hg for 6 hours. The anterior segments were then quickly frozen in liquid nitrogen, and their TMs were dissected for RNA extraction. SMART cDNA libraries were generated from control and high-pressure human TM RNAs and hybridized to sets of identical high-density cDNA gene arrays. These arrays contained 18,376 human expressed sequence tags (ESTs), corresponding to both characterized and unknown genes. Differentially expressed genes were identified by different-intensity hybridization signals and confirmed by semi-quantitative polymerase chain reaction. RESULTS: Eleven genes were found to be consistently upregulated in the human TM by elevated IOP: interleukin-6, preprotachykinin-1, secretogranin-II, cathepsin-L, stromelysin-1, thymosin-beta4, alpha-tubulin, alphaB-crystallin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein and Cu/Zn superoxide dismutase. The products of these genes are involved in vascular permeability, secretion, extracellular matrix remodeling, cytoskeleton reorganization, and reactive oxygen species scavenging. CONCLUSIONS: Elevated IOP induced specific upregulation of 11 physiologically relevant genes. On the basis of their known activities, the products of each of these genes might predict homeostatic mechanisms similar to those involved in the regulation of blood vessel permeability. We hypothesize that similar mechanisms might be involved in regulating flow through Schlemm's Canal endothelium.

Aged↗
PubMed2003

Involvement of AP-1 in interleukin-1alpha-stimulated MMP-3 expression in human trabecular meshwork cells.

PURPOSE: Stromelysin-1 (MMP-3) degrades extracellular matrix and increases aqueous outflow. In the trabecular meshwork (TM), interleukin (IL)-1alpha is a potent inducer of MMP-3 expression. In different cells, IL-1alpha activates different signaling pathways, such as nuclear factor (NF)-kappaB-mediated protein expression, the phospholipase A(2) (PLA(2))-activated arachidonate cascade, and activator protein (AP)-1-associated transcription. In the present study, pharmacological tools were used to delineate the signaling mechanism involved in the effect of IL-1alpha on MMP-3 production in human TM cells compared with other ocular cells. METHODS: Human TM and three other ocular cells (ciliary muscle, corneoscleral fibroblast, and lamina cribrosa) were cultured in 24-well plates in the presence or absence of IL-1alpha, with or without specific inhibitors of selected signaling pathways. Secreted proMMP-3 was quantified by ELISA, and MMP-3 activity was assayed by casein zymography. RESULTS: IL-1alpha (5 ng/mL) increased proMMP-3 levels in human TM cells to 10- to 38-fold of control (P < 0.001). The effect of IL-1alpha was blocked by Gö6976, a protein kinase C micro (PKC micro ) inhibitor; PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor; SB202190, a p38 inhibitor; and SR11302, an AP-1 inhibitor; but not by inhibitors of casein kinase II, NFkappaB, PLA(2), phospholipase D (PLD), cyclooxygenases, lipoxygenase, or sphingomyelinase. SR11302 did not inhibit the effect of IL-1alpha on MMP-3 production in the other ocular cells tested. CONCLUSIONS: Based on the pharmacological effects of the inhibitors, the data indicate that activation of PKC micro, MEK, and p38 leading to the activation of AP-1 is critical to the IL-1alpha-stimulated upregulation of MMP-3 in human TM cells. Therefore, it is likely that compounds that activate the AP-1 pathway would upregulate the production of MMP-3 and improve aqueous outflow.

Adolescent↗
PubMed2006

Detection of differentially expressed glycogenes in trabecular meshwork of eyes with primary open-angle glaucoma.

PURPOSE: To identify differentially expressed glycogenes in trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG). METHODS: Total RNA was isolated from TM of cadaveric eyes derived from donors with diagnosed glaucomas of different etiologies and from normal control subjects. RNA was amplified and hybridized to the GLYCOv2 oligonucleotide microarray that contains probes for carbohydrate-binding proteins, glycosyltransferases, and other genes involved in the regulation of glycosylation. Statistical analysis was used to identify differentially expressed genes between normal and POAG samples. RESULTS: This study revealed that POAG TM and normal TM have distinct gene expression profiles. Of the 2001 genes on the array, 19 genes showed differential expression of greater than 1.4-fold in POAG. Mimecan and activinA, which have been shown to be upregulated in models of glaucoma, were both found to be elevated in POAG TM. Many genes were identified for the first time to be differentially regulated in POAG. Among the upregulated genes were: (1) cell adhesion molecules including platelet endothelial cell adhesion molecule-1 and P-selectin, both of which are targets of NFkappaB, which has been shown to be activated in glaucomatous TM; (2) lumican, a core protein of keratan sulfate proteoglycans; and (3) the receptor for IL6, a cytokine that has been shown to be upregulated in TM in response to elevated intraocular pressure. Among the downregulated genes were chondroitin-4-O-sulfotransferase involved in the synthesis of chondroitin sulfate chains and the receptor for PDGFbeta, a growth factor that has been shown to stimulate both TM cell proliferation and phagocytic activity. Results for several genes were confirmed by RTq-PCR. CONCLUSIONS: Microarray technology was used to show, for the first time, that POAG TM has a distinct glycogene expression profile. Differentially expressed glycogenes identified in this study have not been previously investigated for their role in the pathogenesis of POAG and thus are novel factors for further study of the mechanism of the disease and for their possible use as diagnostic markers.

Aged↗
PubMed1983

Human trabecular meshwork in primary culture: a morphological and autoradiographic study.

The growth and behaviour of human trabecular meshwork cells in primary culture was studied by light microscopy, time-lapse cinephotomicrography, autoradiography and electron microscopy. In all 925 explants of trabecular tissue from 132 eyes (68 +/- 14 years) were set up, and 20% produced extensive monolayers. Failure to produce primary outgrowths did not increase substantially until the eyes were over 5 days post-mortem. The latent period prior to growth was anything from a few days to 4 weeks; only then did meshwork cells label with [3H]-thymidine. The cells which grew incorporated large amounts of [3H]-leucine and this served as a useful marker to identify migratory cells. In addition they had the ultrastructural features of metabolically active cells including many mitochondria, a well-developed Golgi apparatus, abundant rough endoplasmic reticulum and prominent lysosomes. It was considered that the cells in primary culture had many features in common with 'activated' rather than normal meshwork cells.

Adult↗
PubMed1981

Developmental immaturity of the trabecular meshwork in congenital glaucoma.

We examined nine specimens of anterior chamber angle tissue obtained by trabeculectomy from seven patients with early-onset congenital glaucoma and one patient with late-onset congenital glaucoma by light and electron microscopy and compared them with specimens of trabecular meshwork from normal human and monkey eyes. One eye with early-onset congenital glaucoma had no Schlemm's canal. In all cases of congenital glaucoma, we observed a thick subcanalicular tissue with a structure similar to that seen in the endothelial meshwork beneath the inner wall of Schlemm's canal. There were abnormal deposits of ground substances that resembled basement membrane. Additionally, histologic studies of eyes from premature infants obtained at autopsy showed that trabecular sheet-formation initially occurs on the anterior chamber side of the trabeculum and advances gradually toward Schlemm's canal and that the undifferentiated portion of the trabeculum remains as endothelial meshwork beneath the inner wall of Schlemm's canal. Our observations strongly suggest that the existence of the thick subcanalicular tissue, which is considered to be endothelial meshwork, indicates an immature stage of the trabecular meshwork and may be one of the primary causes of increased intraocular pressure in congenital glaucoma.

Animals↗
PubMed2001

Delayed secondary glucocorticoid responsiveness of MYOC in human trabecular meshwork cells.

PURPOSE: To characterize the glucocorticoid responsiveness of the glaucoma gene MYOC (myocilin/TIGR) in cultured human trabecular meshwork (TM) cells. METHODS: MYOC expression in two independently derived human TM cell lines was quantified by Western immunoblot analysis of protein levels and quantitative PCR analysis of mRNA levels. Promoter activity was measured indirectly with the luciferase reporter gene in a dual luciferase reporter assay. RESULTS: Application of the synthetic glucocorticoid dexamethasone (Dex) to cultured TM cells at 100 nM resulted in a delayed (8-16 hours) induction of myocilin. The concentration dependence (median effective concentration [EC(50)], approximately 10 nM) and reversal by the glucocorticoid antagonist, RU486, implicates the glucocorticoid receptor (GR). In an interesting observation, RU486 alone acted as a partial agonist to MYOC expression. Treatment of TM cells with the protein synthesis inhibitor cycloheximide abolished the Dex induction, suggesting an indirect effect of the GR on MYOC expression. In addition, the RNA synthesis inhibitor actinomycin D also blocked Dex induction, indicating that the Dex effect was due to increased MYOC transcription. Analysis of up to 2700 nucleotides (nt) of the MYOC gene 5'-flanking region in luciferase reporter constructs showed no Dex induction, despite the presence of multiple putative glucocorticoid response element (GRE)-like half-sites in the MYOC promoter and the presence of an intact cellular GR-mediated signaling system. CONCLUSIONS: MYOC is a delayed secondary glucocorticoid-responsive gene. Characterization of the transcription factors that mediate the secondary response will shed new light on the pathophysiology of steroid-induced ocular hypertension and glaucoma.

Cells, Cultured↗
PubMed2006

Effect of dexamethasone and aquaporin-1 antisense oligonucleotides on the aquaporin-1 expression in cultured human trabecular meshwork cells.

The changes in the expression of aquaporin-1 (AQP1) mRNA and protein in cultured human trabecular meshwork (HTM) cells treated with dexamethasone and transfected with antisense oligonucleotides (AS-ODN) were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0. 04 microg/mL and 0.4 microg/mL groups, the levels of AQP1 were higher than in control group (0 microg/mL). In the 4 microg/ mL and 40 microg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 microg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Aquaporin 1↗
PubMed2005

Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter.

PURPOSE: To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway. METHODS: The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ. RESULTS: gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments. CONCLUSIONS: Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.

Adenoviridae↗
PubMed1993

[Lectin histochemical studies on species differences in the mammalian trabecular meshwork].

Lectin histochemical studies were performed to clarify the species differences in the location of glycoconjugates in the trabecular meshwork (TM) of various mammals, i.e., the mouse, rat, rabbit, pig, and ox, to find an experimental model for human TM. Cryosections were made and stained with sixteen kinds of biotinylated lectin followed by an avidin-biotin-peroxidase complex (ABC). Strong positive reactions in the TM of all 5 kinds of mammals were observed for ConA, PHA-E4, PHA-L4, WGA, ABA, LCA, RCA 60, RCA 120, DSA, and SSA. The TM of the 5 kinds of mammals was weakly positive for Lotus. Rabbit, pig and ox TM were positive for MAM and others were negative. Rabbit, pig and ox TM were weakly positive for PNA and were negative in the other's. Rat TM was weakly positive for SBA and was negative in the other's. The TM of all 5 kinds of mammals was negative for UEA-I and DBA. It could be concluded that species difference exists in lectin binding site in the TM.

Animals↗
PubMedDate not supplied

Effects of adrenergic agents and phosphodiesterase inhibitors on outflow facility and cell shape of bovine trabecular meshwork.

Changes in the outflow facility of perfused calf eyes and in the shape of cells in cultured trabecular meshwork (TM) have been studied, following exposure to adrenergic agents and phosphodiesterase inhibitors (PDE). Dobutamine caused confluent TM cells to change their usual polygonal shape to a characteristic stellate shape. Salbutamol had no effect, but PDE inhibitors, isobutylmethylxanthine (IBMX), theophylline, and caffeine were very effective in producing this shape change. Epinephrine, isoproterenol, dobutamine, and salbutamol did not increase the outflow facility, either at 22 degrees C or 36 degrees C, while theophylline, caffeine, and IBMX did increase it in a dose-dependent manner. On the other hand, the high concentrations of beta-adrenergic agents required to produce even a small change in outflow facility and cell shape argue against the involvement of adrenergic-receptor mediation and may suggest another mechanism; on the other, the enhancement of epinephrine effects by PDE inhibitors and the similar effect produced by cyclic adenosine 3',5'-cyclic phosphate (cAMP) and purines suggest that changes in the cell shape are produced by beta-receptor activation. The beta-adrenergic agents were not effective in changing outflow facility, but the PDE inhibitors were remarkably effective both in changing the shape and in increasing facility.

Administration, Topical↗
PubMed2000

Signal transduction mediated by adhesion of human trabecular meshwork cells to extracellular matrix.

In this study we investigated the signaling event induced by adhesion of human trabecular meshwork (TM) cells to extracellular matrix (ECM) elements such as fibronectin. The role of tyrosine phosphorylation in adhesion was evaluated. A number of intracellular entities involved in the adhesion-mediated pathways were identified. For the experiments, human TM cells were seeded onto fibronectin- or polylysine (negative control)-coated plates. Fifteen, 30, 90 and 240 min after the seeding, cell lysates were collected. Immunoblotting analysis revealed that tyrosine phosphorylation occurred within 15 min of adhesion of TM cells to fibronectin and the level increased with time. The phosphotyrosyl proteins had molecular masses 25-220 kDa. A much lower level of tyrosine phosphorylation was observed when cells were plated on polylysine. Immunoprecipitation experiments indicated that the phosphotyrosine-containing proteins included focal adhesion kinase, paxillin, phosphatidylinositol 3-kinase and mitogen activated protein kinase. Within 30 min of adherence to fibronectin, human TM cells immunostained for paxillin and phosphotyrosine and exhibited prominent focal contacts. When treated with tyrosine kinase inhibitors genistein and herbimycin A and a protein kinase C (PKC) pseudosubstrate peptide inhibitor, cell adhesion to fibronectin was compromised and focal contact formation was limited. These results demonstrated that in human TM cells, tyrosine kinase was activated upon their adherence to fibronectin. PKC also appeared to play a role in modulation of the cell-matrix adhesion process. The current study provides insight into the signaling pathways that are linked to the ECM-induced events in TM cells. Elucidation of the hierarchy of signal responses may help develop strategies manipulating the cell-matrix interactions in the TM system.

Adult↗
PubMed2005

Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility.

Cellular contraction and relaxation and integrity of the actin cytoskeleton in trabecular meshwork (TM) tissue have been thought to influence aqueous humour outflow. However, the cellular pathways that regulate these events in TM cells are not well understood. In this study, we investigated physiological agonist-mediated regulation of myosin light chain (MLC) phosphorylation in the TM, and correlated such effects with alterations in aqueous outflow facility, since MLC phosphorylation is a critical biochemical determinant of cellular contraction in TM cells. Treatment of serum starved human TM cells with endothelin-1 (0.1 microM), thromboxane A2 mimetic U-46619 (1.0 microM), or angiotensin II (1 microM), all of which are agonists of G-protein coupled receptors, triggered activation of MLC phosphorylation, as determined by urea/glycerol-based Western blot analysis. Agonist-stimulated increase in MLC phosphorylation was associated with activation of Rho GTPase in TM cells, as determined in pull-down assays. In contrast, treatment of human TM cells with a novel Rho-kinase inhibitor H-1152 (0.1-2 microM), in the presence of serum reduced basal MLC phosphorylation. H-1152 also increased aqueous outflow facility significantly in a dose-dependent fashion, in perfusion studies with cadaver porcine eyes. This effect of H-1152 on outflow facility was associated with decreased MLC phosphorylation in TM tissue of drug-perfused eyes. Collectively, this study identifies potential physiological regulators of MLC phosphorylation in human TM cells and demonstrates the significance of Rho/Rho-kinase pathway-mediated MLC phosphorylation in modulation of aqueous outflow facility through TM.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Immunomicroscopical study of type VI collagen in the trabecular meshwork of normal and glaucomatous eyes.

Cross-strained fiber bundles called long-spacing collagen or curly collagen occur in normal eyes in the trabecular meshwork. It can be seen in the basement membrane of the trabecular lamellae, in the sheath of the elastic-like fibers and underneath the inner wall of Schlemm's canal, where it forms part of the so called plaque material. The amount of this long-spacing collagen increases with age and is significantly more pronounced in glaucomatous eyes. Using immunohistochemical and immuno-electronmicroscopic methods, we have been able to show that type VI collagen is present in the aggregates called long-spacing collagen.

Age Factors↗
PubMed2006

IL-1 and TNF induction of matrix metalloproteinase-3 by c-Jun N-terminal kinase in trabecular meshwork.

PURPOSE: The cytokines TNF and IL-1 mediate the MMP-3 increase that occurs in response to trabecular meshwork (TM) treatment by laser trabeculoplasty. This MMP-3 increase appears to play a key role in the efficacy of this treatment for open-angle glaucoma. Protein kinase Cmu and the Erk mitogen-activated protein (MAP) kinases are essential signaling components in transducing MMP-3 increases produced by treatment of TM cells with these cytokines. Here, the involvement of the JNK-MAP kinase pathway in this process was evaluated. METHODS: Porcine TM cells were treated with TNFalpha, IL-1alpha, or IL-1beta. Changes in MMP-3 and MMP-9 protein levels in the media were then determined by Western immunoblot. The effect of JNK inhibitor 2 was evaluated. Changes in the level of phosphorylation of JNK, c-Jun, ATF-2, MKK4, and MKK7 were also determined at various times after TNFalpha or IL-1alpha treatment. A 2.3-kb MMP-3 promoter fragment was cloned into a secreted alkaline phosphatase reporter vector. This reporter construct was cotransfected into TM cells with a mammalian expression vector containing a dominant-negative mutant of JNK. The involvement of JNK activity in the TNFalpha and IL-1alpha induction of MMP-3 expression was then evaluated. RESULTS: TNFalpha, IL-1alpha, and IL-1beta increase media MMP-3 and MMP-9 protein levels, and JNK inhibitor 2 blocks these increases. JNK1/2, MKK4, c-Jun, and ATF-2 phosphorylation levels increase in response to TNFalpha and IL-1alpha treatment. JNK inhibitor 2 pretreatment blocks these c-Jun and ATF-2 phosphorylation increases. Dominant-negative JNK dramatically reduces the MMP-3 promoter-driven reporter activity induced by these cytokines. CONCLUSIONS: JNK activity is necessary for the induction of MMP-3 and MMP-9 by TNFalpha, IL-1alpha, or IL-1beta in TM cells. Phosphorylation of components of the JNK signaling pathway and of the transcription factors c-Jun and ATF-2 support a role for this pathway in the induction of MMP-3 and MMP-9 in the TM in response to these cytokines. Thus, at least three separate signal transduction pathways are necessary in this signaling event in TM cells.

Activating Transcription Factor 2↗