Lactate treatment causes NF-kappaB activation and CD44 shedding in cultured trabecular meshwork cells.
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.
Miller
Adam M
AM
Laboratory for Oculo-Cerebrospinal Investigation, Division of Neurosurgery, Children's Memorial Medical Center, Chicago, Illinois, USA.
Nolan
Michael J
MJ
Choi
John
J
Koga
Tomoyo
T
Shen
Xiang
X
Yue
Beatrice Y J T
BY
Knepper
Paul A
PA
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*
C497483:CD44 protein, human; D018960:Hyaluronan Receptors; D051996:Transcription Factor RelA; D019344:Lactic Acid; D053511:Matrix Metalloproteinase 14