title: Lactate treatment causes NF-kappaB activation and CD44 shedding in cultured trabecular meshwork cells.
abstract: OBJECTIVE
  To challenge human trabecular meshwork (TM) cells using lactate to mimic cell stress and observe the effects on cell viability, NF-kappaB, and membrane type 1 matrix metalloproteinase (MT1-MMP) expression and the ectodomain shedding of soluble (s)CD44.
  
  
  METHODS
  Human TM cells grown in 10% fetal calf serum (FCS) were incubated in 0.1% FCS with 1, 10, or 40 mM lactate or PBS for 5 and 30 minutes and 1, 3, and 6 hours. Cell viability was determined with trypan blue staining. NF-kappaB and MT1-MMP expression was evaluated through Western blot analysis of medium and the cytoplasmic and nuclear fractions. Media sCD44 concentration was determined by enzyme-linked immunosorbent assay and Western blot analysis.
  
  
  RESULTS
  The TM cell viability was significantly decreased after incubation for 3 hours with 40 mM lactate (P < 0.01) and 6 hours with 10 and 40 mM lactate (P < 0.001). Western blot analysis showed an increased NF-kappaB p50 and MT1-MMP expression and activity by 5 minutes in lactate-treated TM cells compared with that of control cells. At 6 hours, NF-kappaB p65 was increased in nuclear fraction of lactate-treated compared with control cells. Treatment with 1 mM lactate caused an increase in the media concentration of both the 32 and 55 kDa sCD44 at 3 (P < 0.05) and 6 hours (P < 0.01).
  
  
  CONCLUSIONS
  Lactate treatment resulted in dose- and time-dependent effects on human TM cell viability, translocation of NF-kappaB, and activation of MT1-MMP. Increased shedding of sCD44 occurred with the l mM dose of lactate. Lactate treatment of human TM cells in culture offers a useful cell model to examine the stress responses that occur in glaucoma.
authors: Laboratory for Oculo-Cerebrospinal Investigation, Division of Neurosurgery, Children's Memorial Medical Center, Chicago, Illinois, USA.
authors: Adam M
authors: AM
authors: Miller
authors: Michael J
authors: MJ
authors: Nolan
authors: John
authors: J
authors: Choi
authors: Tomoyo
authors: T
authors: Koga
authors: Xiang
authors: X
authors: Shen
authors: Beatrice Y J T
authors: BY
authors: Yue
authors: Paul A
authors: PA
authors: Knepper
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D019344:Lactic Acid / Q000494:pharmacology*; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D008875:Middle Aged; D013997:Time Factors; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: C497483:CD44 protein, human; D018960:Hyaluronan Receptors; D051996:Transcription Factor RelA; D019344:Lactic Acid; D053511:Matrix Metalloproteinase 14