Regulated expression of caspase-12 gene in human retinal pigment epithelial cells suggests its immunomodulating role.
OBJECTIVE
To investigate the expression and regulation of the short form of caspase-12, caspase-12S, in human retinal pigment epithelial (hRPE) cells.


METHODS
hRPE cells were stimulated by the proinflammatory agents IL-1beta (2 ng/mL) and TNF-alpha (20 ng/mL); LPS (1000 ng/mL); coculture with monocytes; the immunomodulating agent cyclosporine (Cys; 30 ng/mL); the anti-inflammatory cytokine IL-10 (100 U/mL); and the endoplasmic reticulum (ER) stress inducers tunicamycin (3 or 10 muM) and thapsigargin (25 or 100 nM) for 6 hours or longer. The total RNAs were isolated and subjected to semiquantitative and quantitative real-time RT-PCR analysis. Effects of tunicamycin and thapsigargin on IL-1beta- and TNF-alpha-stimulated MCP-1 mRNA expression and protein production were further examined by RT-PCR and ELISA, respectively.


RESULTS
RT-PCR results showed that caspase-12S is the predominant form of caspase-12 in the examined hRPE cells of this study, with expression at levels as high as those in many other human tissues such as pancreas, prostate, small intestine, lung, spleen, and kidney. Treatment with IL-1beta and TNF-alpha, as well as LPS and coculture with monocytes reduced hRPE caspase-12S mRNA expression within 6 hours. In contrast, hRPE exposure to cyclosporine (Cys) and the cytokine IL-10 for 6 hours increased caspase-12S mRNA expression. Compared to Cys and IL-10, the ER stress activators tunicamycin and thapsigargin were even more potent enhancers of hRPE caspase-12S gene expression. They also caused corresponding reductions in IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression and protein production.


CONCLUSIONS
hRPE cells express a high level of caspase-12S. The regulated expression of caspase-12S suggests that this caspase recruitment domain (CARD)-only protein may be an endogenous dominant negative regulator that modulates inflammatory responses in hRPE cells.
Bian
Zong-Mei
ZM
Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan 48105, USA.
Elner
Susan G
SG
Elner
Victor M
VM
D017209:Apoptosis; D053454:Caspase 12 / Q000235:genetics*; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D016572:Cyclosporine / Q000494:pharmacology; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019284:Thapsigargin / Q000494:pharmacology; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014415:Tunicamycin; D016753:Interleukin-10; D019284:Thapsigargin; D016572:Cyclosporine; C506444:CASP12 protein, human; D053454:Caspase 12
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