Up‐regulation of PYK2/PKCα‐dependent haem oxygenase‐1 by CO‐releasing molecule‐2 attenuates TNF‐α‐induced lung inflammation

Background and Purpose Haem oxygenase‐1 (HO‐1) could provide cytoprotection against various inflammatory diseases. However, the mechanisms underlying the protective effect of CO‐releasing molecule‐2 (CORM‐2)‐induced HO‐1 expression against TNF‐α‐induced inflammatory responses in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unknown. Experimental Approach CORM‐2‐induced HO‐1 protein and mRNA expression, and signalling pathways were determined by Western blot and real‐time PCR, coupled with respective pharmacological inhibitors or transfection with siRNAs. The effect of CORM‐2 on TNF‐α‐induced increase in leukocyte counts in BAL fluid and VCAM‐1 expression in lung was determined by cell counting and Western blot analysis. Key Results CORM‐2 attenuated the TNF‐α‐induced pulmonary haematoma, VCAM‐1 expression and increase in leukocytes through an up‐regulation of HO‐1 in mice; this effect of CORM‐2 was reversed by the HO‐1 inhibitor zinc protoporphyrin IX. Furthermore, CORM‐2 increased HO‐1 protein and mRNA expression as well as the phosphorylation of PYK2, PKCα and ERK1/2 (p44/p42 MAPK) in HPAEpiCs; these effects were attenuated by their respective pharmacological inhibitors or transfection with siRNAs. Inhibition of PKCα by Gö6976 or Gö6983 attenuated CORM‐2‐induced stimulation of PKCα and ERK1/2 phosphorylation but had no effect on PYK2 phosphorylation. Moreover, inhibition of PYK2 by PF431396 reduced the phosphorylation of all three protein kinases. Finally, PYK2/PKCα/ERK1/2‐mediated stimulation of activator protein 1 was shown to play a key role in CORM‐2‐induced HO‐1 expression via an up‐regulation of c‐Fos mRNA. Conclusions and Implications CORM‐2 activates a PYK2/PKCα/ERK1/2/AP‐1 pathway leading to HO‐1 expression in HPAEpiCs. This HO‐1/CO system might have potential as a therapeutic target in pulmonary inflammation.