title: Synergism of TNF and IL-1 in the induction of matrix metalloproteinase-3 in trabecular meshwork.
abstract: OBJECTIVE
  TNF and IL-1 increase matrix metalloproteinase-3 (MMP-3) expression in the trabecular meshwork (TM). TNF-alpha, in combination with IL-1alpha or IL-1beta, produces highly synergistic MMP-3 increases. Possible mechanisms for this synergism in TM cells were investigated.
  
  
  METHODS
  Porcine and human TM cells were treated with TNF-alpha, IL-1alpha, IL-1beta and their combinations. Western immunoblots were used to evaluate MMP-3, MMP-9, MMP-12, TNF-alpha, IL-1alpha, IL-1beta, IL-6, TNF receptor I (RI), IL-1 RI, and IL-1 RII levels and the phosphorylation of Erk, JNK, and p38 MAP kinases. Dose-response effects for TNF-alpha, IL-1alpha and IL-1beta on MMP-3 were evaluated. Microarray and quantitative RT-PCR were used to determine mRNA levels. MMP-3 transcription rate was assessed by transfecting TM cells with an MMP-3 promoter/reporter construct. Combined cytokine effects on outflow facility were appraised in perfused anterior segment organ culture.
  
  
  RESULTS
  TNF-alpha, IL-1alpha, and IL-1beta each individually increased MMP-3 levels, whereas TNF-alpha in combination with IL-1alpha or IL-1beta produced highly synergistic increases. MMP-9 and MMP-12 levels were also elevated, but only MMP-12 showed synergism. IL-1alpha, IL-1beta, and IL-6, but not TNF-alpha mRNA or protein level, were elevated by these cytokines. Maximum MMP-3 production for individual cytokines, even at high doses, was far less than with dual cytokine doses. Erk 1 and 2, JNK 1 and 2, and p38 alpha and beta phosphorylation increased, but not synergistically. However, phosphorylation of novel isoforms of JNK and p38 delta and gamma did show synergism. MMP-3 mRNA levels and transcription rates also demonstrated synergism. TNF-alpha significantly increased IL-1 RI levels. Synergism in outflow facility was observed with TNF-alpha and IL-1alpha.
  
  
  CONCLUSIONS
  TNF-alpha, in combination with IL-1alpha or IL-1beta, produced intense synergistic increases in MMP-3 and MMP-12 but not in MMP-9. Induction of IL-1 RI by TNF-alpha partially explains the synergism. Responses of novel JNK and p38 MAP kinase delta and gamma isoforms also partially account for the synergism. Understanding this strong synergistic effect may provide useful insight into optimizing therapeutic regulation of intraocular pressure in glaucoma.
authors.affiliation: Casey Eye Institute, Oregon Health and Science University, Portland, Oregon 97239-4197, USA.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Kelley
authors.forename: Anastasia Y
authors.initials: AY
authors.lastname: Rose
authors.forename: Kaili
authors.initials: K
authors.lastname: Song
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Chen
authors.forename: John M
authors.initials: JM
authors.lastname: Bradley
authors.forename: Derek
authors.initials: D
authors.lastname: Rookhuizen
authors.forename: Ted S
authors.initials: TS
authors.lastname: Acott
mesh_terms: D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000378:metabolism; D053582:Interleukin-1alpha / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D053508:Matrix Metalloproteinase 12 / Q000096:biosynthesis; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D053590:Interleukin 1 Receptor Antagonist Protein; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3; D053508:Matrix Metalloproteinase 12