Contribution of calpain to cellular damage in human retinal pigment epithelial cells cultured with zinc chelator.
OBJECTIVE
We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD.


METHODS
In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenase (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate alpha -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium.


RESULTS
RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and alpha -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE.


CONCLUSIONS
These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.
Tamada
Yoshiyuki
Y
Senju Laboratory of Ocular Sciences, Beaverton, Oregon 97006, USA.
Walkup
Ryan D
RD
Shearer
Thomas R
TR
Azuma
Mitsuyoshi
M
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D009842:Oligopeptides / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013049:Spectrin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D061945:Caspase Inhibitors; D002614:Chelating Agents; D004151:Dipeptides; D005029:Ethylenediamines; C109145:N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal; D009842:Oligopeptides; D012333:RNA, Messenger; C120992:benzyloxycarbonyl aspartyl-glutamyl-valyl-aspartyl-chloromethyl ketone; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; D013049:Spectrin; D007770:L-Lactate Dehydrogenase; D002154:Calpain; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium