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Regulation of calpain and calpastatin by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1) in murine aortic endothelial cells
Author(s) -
Liu Tongzheng,
Likhotvorik Rostislav,
Huang Yongcheng,
Keshvara Lakhu,
Hoyt Dale G
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.964.35
Subject(s) - calpastatin , calpain , pin1 , small hairpin rna , microbiology and biotechnology , transfection , gene knockdown , chemistry , cypa , peptidylprolyl isomerase , cyclophilin a , biology , gene , biochemistry , isomerase , enzyme
The peptidyl‐proline isomerase, PIN1, regulates protein turnover and function. We found that PIN1 normally restrains the induction of iNOS by facilitating calpain‐mediated degradation in murine aortic endothelial cells (EC), suggesting that PIN1 facilitates calpain activity in EC. To test this theory, EC were stably transfected with a vector producing a PIN1 short hairpin RNA (shRNA) or an inactive control sequence. PIN1 shRNA reduced PIN1 levels >85% compared to control. Calpain activity in cell extracts was measured with a fluorescently quenched substrate, (DABCYL)‐TPLK~SPPPSPR‐(EDANS). Incubation of control EC with 10 μg LPS and 20 ng IFN for 24 h increased the initial velocity of cleavage by >5.8 fold. Activity in EC lacking PIN1 was only 45% of the control cells, with or without LPS/IFN treatment. However, there was no reduction of Calpain‐1 or ‐2 protein levels by western blotting. Instead, calpastatin, an endogenous protein inhibitor of Calpain‐1 and ‐2, was increased >2.5‐fold in PIN knockdown cells. PIN1 depletion did not affect calpastatin mRNA levels, suggesting a post‐transcriptional effect. The results suggest that PIN1 normally enhances calpain activity by post‐transcriptional suppression of calpastatin. Alterations in PIN1 expression or function may significantly affect a broad range of calpain‐dependent processes, particularly in EC.