c-Src-Dependent Transactivation of EGFR Mediates CORM-2-Induced HO-1 Expression in Human Tracheal Smooth Muscle Cells CHUEN-MAO YANG, 1 * CHIH-CHUNG LIN, 2 I-TA LEE, 1 CHIH-KAI HSU, 1 YU-CHEN TAI, 1 HSI-LUNG HSIEH, 3 PEI-LING CHI, 1 AND LI-DER HSIAO 2 1 Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan 2 Department of Anesthetics, Chang Gung Memorial Hospital at Linkuo and Chang Gung University, Kwei-San, Tao-Yuan, Taiwan 3 Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Kwei-San, Tao-Yuan, Taiwan Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, displays an anti-inflammatory effect in various cellular injuries, but the precise mechanisms of HO-1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 (CORM-2) that acts as carbon monoxide donor. The effects of CORM-2 on expression of HO-1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real-time PCR, and promoter activity assay. In HTSMCs, CORM-2 activated Nrf2 through the activation of a c-Src/EGFR/PI3K/Akt-dependent pathway, resulting in HO-1 expression. We showed that CORM-2-induced HO-1 protein and mRNA levels were inhibited by the inhibitor of c-Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c-Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM-2 stimulated c-Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM-2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM-2 mediated p38 MAPK and JNK1/2 activation via a c-Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM-2 induced in vivo binding of Nrf2 to the HO-1 promoter. CORM-2 activates the c-Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO-1 expression in HTSMCs. Thus, the HO-1/CO system might be potential therapeutics in airway diseases. J. Cell. Physiol. 230: 2351–2361, 2015. © 2015 Wiley Periodicals, Inc. It is established that tracheal smooth muscle has roles in determining airway structure and function, well beyond that as the major contractile element. Moreover, changes in tracheal smooth muscle function are central to the manifestation of allergic, inflammatory, and fibrotic airway diseases, as well as to airway responses to local and environmental exposures (Lee et al., 2010). Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step of heme degradation, resulting in the formation of iron, carbon monoxide (CO), and biliverdin, which is subsequently converted to bilirubin by biliverdin reductase. Recent attention has focused on the biological effects of product(s) of this enzymatic reaction, which have important antioxidant, anti-inflammatory, and cytoprotective functions (Lee et al., 2009). Induction of HO-1 occurs as an adaptive and beneficial response to several injurious stimuli, and has been implicated in many clinically relevant disease states (Lee et al., 2008). Since CO inhalation cannot be accurately regulated and may be toxic to tissues with prolonged exposure, CO-releasing molecules (CORMs) were developed to allow selective delivery and local release of CO from a nontoxic pro-drug. A transition metal carbonyl-based compound, CORM-2 can efficiently release controlled amounts of CO. In addition, CORM-2 treatment is associated with minimal formation of carboxyhemoglobin, suggesting that CORM-2 is a safer alternative than inhaled CO (Constantin et al., 2012). The Conflict of interest: The authors declared that they have no competing interests. Contract grant sponsor: Ministry of Science and Technology, Taiwan; Contract grant numbers: MOST102-2321-B-182-011, MOST101- 2320-B-182-039-MY3, MOST101-2314-B-182-182A-112. Contract grant sponsor: Chang Gung Medical Research Foundation; Contract grant numbers: CMRPD1C0102, CMRPD1B0382, CMRPD1B0383, CMRPD1C0562, CMRPG3B1093, CMRPG3C1302. Contract grant sponsor: Ministry of Education, Taiwan; Contract grant number: EMRPD1E1641. *Correspondence to: Chuen-Mao Yang, Department of Pharmacology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan. E-mail: chuenmao@mail.cgu.edu.tw Manuscript Received: 6 February 2014 Manuscript Accepted: 18 December 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 29 April 2015. DOI: 10.1002/jcp.24912 ORIGINAL RESEARCH ARTICLE 2351 Journal of Journal of Cellular Physiology Cellular Physiology © 2015 WILEY PERIODICALS, INC.