200 Fig. 2. H.-O. Pae et al. Potential signaling pathways activated by HO-1 and heme metabolites leading to tissue protection. HO-1 reduces the pro- oxidant heme through enzymatic reaction. Iron released from this reaction triggers pathways involving the activation of the iron regulatory protein, leading to its sequestration and recycle. BR and BV potentially contribute to cellular antioxidant balance prior to their excretion. CO activates sGC, resulting in production of cGMP. CO also modulates MAPK pathways, thereby leading to anti-inflammatory, anti-apoptotic, and antiproliferative effects. The solid and dotted arrow lines shows either an increase or activation and either a decrease or inactivation, respectively. over, it is becoming clear that low plasma concentrations of BR are associated with a low incidence of cardiovascular disorders in humans (e.g., atherosclerosis and ischemic heart disease) [36]. HO-1 Inducer As HO-1 expression is widely recognized as an effective cellular strategy to counteract a variety of stressful events [4], the induction of HO-1 by pharmacological modulators may represent a novel target for therapeutic intervention. Particularly, the identification of non-cytotoxic HO-1 inducers may maximize the intrinsic protective potential of cells. Phytochemicals Several antioxidants from plant origins and drugs have been reported to induce HO-1 expression in various types of cells [37]. Among them, curcumin (Fig. 3A), a polyphenolic compound isolated from the rhizome of Curcuma longa Linn (Zingiberaceae), is well characterized as a non- cytotoxic HO-1 inducer in endothelial cells [16], astrocytes [38], macrophages [39], and vascular smooth muscle cells [40]. In the liver HepG2 cells, curcumin also induced HO-1 expression in a time- and dose-dependent manner (Fig. 3B). Curcumin activated Nrf2 nuclear translocation (Fig. 3C), which is presumably an upstream step of HO-1 expression. The experiments designed to determine a possible role of MAPK pathways in HO-1 expression by curcumin showed that curcumin activates ERK and p38 MAPK pathways (Fig. 3D), but not JNK pathway. Additionally, the use of specific inhibitors for the MAPK pathways confirmed the involvement of p38 MAPK pathway, but not of ERK or JNK pathways, in HO-1 expression (Fig. 3E). Interestingly, pre- incubation for 12 h with curcumin resulted in an enhanced cellular resistance to the potent oxidant t-butylhydro- peroxide; this cytoprotective effect was considerably attenuated by zinc protoporphyrin IX, an inhibitor of HO activity (Fig. 3F). CO Surprisingly, CO can induce HO-1 expression, raising a possibility that CO may exert full effects not only through the first pathway associated with its original ability but also through the second pathway associated with HO-1 expres- sion that, in addition to CO-mediated effects, may provide other effects. In this regard, CO may serve as an effective HO-1 inducer. Sawle and coworkers [41] originally demon- strated that a CO-releasing molecule (CORM) increased HO-1 expression and HO activity. However, they did not explore the cellular mechanism for this effect of CO on HO- 1 expression, as well as its physiological significance. Later, Lee and coworkers [42] confirmed that both CO gas and CORM induced HO-1 expression via Nrf2-dependent pathway in HepG2 cells and primary rat hepatocytes. This is further confirmed by Kim and coworkers [43]. They demon- strated that exogenous CO activated Nrf2 through the phos- phorylation of protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in HO-1 expression. CO-induced activation of PERK was followed by the phosphorylation of eukaryotic translation initiation factor 2 and the expression of activating transcription factor 4. However, CO failed to J. Clin. Biochem. Nutr.