title: HGF signaling impacts severity of Pseudomonas aeruginosa keratitis.
abstract: OBJECTIVE
  To determine whether rapamycin altered corneal growth factor levels to impact severity of Pseudomonas aeruginosa keratitis.
  
  
  METHODS
  BALB/c mice were injected intraperitoneally with rapamycin or PBS and infected with P. aeruginosa. Corneas were harvested and mRNA levels of growth factors (EGF, HGF, FGF-7/KGF), receptors (EGFR, c-met, FGFR-2), and signaling molecules (PI3K, Akt, S6K1, and IGF-1R) tested. ELISA determined HGF/c-met, IGF-1, and Substance P (SP) protein levels. Corneal application of recombinant (r)HGF was assessed by clinical score, photography with a slit lamp, real-time RT-PCR (mRNA for mT0R, IL-10, IL-12, IL-18, PI3KCα, Akt), and ELISA (total and phosphorylated [p]c-met); rIGF-1 effects also were tested by ELISA. In vitro, RAW cells and peritoneal macrophages were stimulated with LPS ± rHGF ± c-met inhibitor (CI) and mTOR mRNA levels tested.
  
  
  RESULTS
  Rapamycin disparately regulated infected corneal mRNA levels of EGF/EGFR and FGF-7/FGFR-2, but HGF/c-met mRNA levels both increased. ELISA confirmed elevated HGF protein. Rapamycin did not change PI3KCα or Akt signaling molecule expression, downregulated S6K1, but upregulated IGF-1R mRNA levels; IGF-1 and SP proteins also were upregulated. After infection, topical rHGF versus PBS increased mRNA levels of IL-12p40, IL-18, PI3KCα, and Akt; mTOR and IL-10 mRNA were downregulated; rIGF-1 increased HGF protein. In vitro, rHGF and LPS lowered RAW cell and macrophage mTOR levels; CI addition restored them.
  
  
  CONCLUSIONS
  Collectively, these data provide evidence that enhanced corneal HGF levels increase signaling through the c-met receptor, decrease mTOR levels, and enhance proinflammatory cytokines, while decreasing anti-inflammatory cytokines, and that HGF signaling is central to disease outcome.
authors.affiliation: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Jiang
authors.forename: Sharon A
authors.initials: SA
authors.lastname: McClellan
authors.forename: Ronald
authors.initials: R
authors.lastname: Barrett
authors.forename: Megan
authors.initials: M
authors.lastname: Foldenauer
authors.forename: Linda D
authors.initials: LD
authors.lastname: Hazlett
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D015818:Eye Infections, Bacterial / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005260:Female; D005786:Gene Expression Regulation; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000378:metabolism; D007634:Keratitis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D011552:Pseudomonas Infections / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012720:Severity of Illness Index; D015398:Signal Transduction
chemical_list: C553307:HGF protein, mouse; D012333:RNA, Messenger; D017228:Hepatocyte Growth Factor
keywords: HGF; IGF-1R; bacteria; c-met; keratitis; mice