Pressure-induced regulation of IL-6 in retinal glial cells: involvement of the ubiquitin/proteasome pathway and NFkappaB.
OBJECTIVE
To investigate how hydrostatic pressure influences regulation of interleukin (IL)-6 by retinal glia and whether this regulation is associated with the ubiquitin/proteasome pathway (UPP) and activation of the transcription factor nuclear factor (NF)kappaB.


METHODS
Astrocytes and microglia isolated from rat retina were maintained in vitro, and the IL-6 concentration in the media at ambient and elevated pressure were compared, with and without the proteasome inhibitor MG132 (10 microM). Immunocytochemistry was used to correlate translocation of NFkappaB with pressure.


RESULTS
Exposure to elevated pressure for 24 hours maximally altered the concentration of media IL-6 of glia cultures, where IL-6 concentrations decreased in astrocyte cultures and increased in microglia cultures. These pressure-induced changes in IL-6 were largely insensitive to MG132 in astrocytes, but were largely MG132-sensitive in microglia. Like IL-6 regulation, pressure-induced activation of NFkappaB also differed between the two glial cell types, where nuclear localization of NFkappaB was transient in astrocytes, but sustained in microglia. Elevated pressure also increased MG132-sensitive expression of IL-6 mRNA by microglia.


CONCLUSIONS
Though pressure-induced regulation of IL-6 by astrocytes is preceded by NFkappaB translocation, it is not altered by MG132 and therefore is not likely to be regulated by NFkappaB or the UPP. In contrast, pressure-induced regulation of IL-6 protein and mRNA by microglia is preceded by NFkappaB translocation and is sensitive to MG132. Together with precedence in the literature, these data suggest that pressure-induced activation of the UPP leads to transcription of IL-6 driven by NFkappaB.
Sappington
Rebecca M
RM
Department of Ophthalmology and Visual Sciences, The Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0654, USA.
Calkins
David J
DJ
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006874:Hydrostatic Pressure; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D015850:Interleukin-6; D007976:Leupeptins; D016328:NF-kappa B; D012333:RNA, Messenger; D043743:Ubiquitin-Protein Ligase Complexes; D013755:Tetradecanoylphorbol Acetate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde