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.