Heme Oxygenase: Mechanisms and Interactions

Heme Degradation

 

Heme oxygenases (HOs) are ER-associated oxygen oxidoreductases involved in the oxidative degradation of heme b. HOs catalyze the cleavage of heme at the α-methene bridge carbon leading to the release of carbon monoxide (CO), and equimolar formation of the open-chain tetrapyrrole biliverdin-IXα (BV), and ferrous iron (Fe2+) 1. The reaction encompasses three oxidation steps (Figure 7), each of which requiring O2 and reducing equivalents provided by NADPH: cytochrome p450 reductase (CPR) 292, 293. Heme not only serves as a substrate but also as a prosthetic group that serially uses a total of three O2 molecules and seven electrons during the oxidation process.

 

The first step, aiming at converting heme to α-meso-hydroxyheme, requires reducing equivalents supplied by CPR for the reduction of the heme iron to the ferrous (Fe2+) state, oxygen binding to the reduced iron, followed by a second one-electron reduction of the oxy-ferrous complex 294. The resulting ferric hydroperoxide (Fe3+-OOH) intermediate functions as an electrophile and hydroxylates the heme ring at the a-methene bridge carbon by inserting the terminal oxygen of the peroxide moiety 295, thereby producing α-meso-hydroxyheme 296, 297.

 

The second step of the catalytic process represents the oxygen-dependent fragmentation of the resulting α-meso-hydroxyheme product to enzyme-bound a-verdoheme. Herein, one molecule of molecular oxygen is used for the displacement of the α-meso-carbon and the bound hydroxyl and their release as CO 298, accompanied by substitution of the carbon in the ring structure by atomic oxygen 299, 300. The implementation and number of reducing equivalents required are still a matter of debate. While the reaction of the ferric α-meso-hydroxyheme with O2 to Fe3+-biliverdin via Fe2+-verdoheme was noted to occur, on the one hand, without the participation of reducing equivalents 301, others postulated the necessity of molecular oxygen and one reducing equivalent for the formation of Fe2+-verdoheme from the α-meso-hydroxyheme 302. In line with this notion, Liu and colleagues found out that oxygen alone is sufficient for the conversion of α-meso-hydroxyheme to Fe3+-verdoheme, even though one electron is necessary for the reduction of the Fe3+-verdoheme complex to the ferrous (Fe2+) state 303.

 

The third step involves the conversion of α-verdoheme to the Fe3+-biliverdin chelate complex, for which O2 and five reduction equivalents from CPR are required. Subsequently, CPR-mediated univalent reduction of Fe3+ in the biliverdin-iron chelate complex induces its dissociation culminating in the release of ferrous ion (Fe2+) and biliverdin IXa 304. Heme oxygenases exhibit a clear preference for heme b; however, a moderate affinity towards heme c and heme a can also be seen 305, 306. Finally, biliverdin is converted to bilirubin IXa by NADP(H):biliverdin reductase (BVR) 1, 29. In the liver, the carboxyl groups in bilirubin IXa are subsequently conjugated with glucuronic acid by bilirubin uridine diphosphate-glucuronosyltransferase 1 (UDPGT-1), thereby increasing its aqueous solubility in preparation for excretion in the urine and bile 307.

 

Ferroptosis

 

Ferroptosis is a recently-recognized type of cell death caused by an accumulation of lipid reactive oxygen species (ROS) leading to iron agglomeration and glutathione (GSH) deficiency 308. Ferroptosis is distinct from classically apoptotic and necrotic cell death types, autophagy and other forms of cell death 308. Cells undergoing ferroptosis show characteristic morphological and biochemical features such as excessive lipid peroxidation, disintegration of mitochondria, and inactivation of cellular GSH-dependent anti-oxidant defense mechanisms 308. Ferroptosis can be induced by a broad spectrum of cellular and pharmacological triggers, including excessive iron loading, inhibition of glutathione peroxidase 4 (GPx4) and the GSH/glutamine antiporter system Xc as well as iron chelating and blockage of ROS formation and lipid peroxidation 308-310. HO-1 which is critically involved in iron and ROS homeostasis shows an ambivalent function in ferroptosis, beneficial and detrimental (for a review, see Chiang et al., 2018) 311. The pro-oxidant activity of HO-1 has been found to induce ferroptosis due to iron accumulation 312-314. Pro-oxidant conditions enhance iron release from iron-storing molecules and thus boost ROS production and oxidative stress, suggesting that a balanced HO-1 activation exerts cytoprotective effects, while hyperactive HO-1 mediates cytotoxicity as a result of an excessive rise in labile Fe2+ behind the scavenging capacity of ferritin 312, 315. Excessive iron release and concomitant ROS production have been found to cause massive oxidative cell damage which culminates in lipid/protein peroxidation and ferroptosis induction 316.

 

Various small molecules have been reported to induce ferroptosis through the modulation of HO-1 expression and activity (Figure 8). The majority of them triggers iron release and the excessive production of ROS. Molecules able to induce HO-1-associated ferroptosis encompass heme 317, magnesium isoglycyrrhizinate 318, BAY117085 313, withaferin A 312 as well as the Xc system inhibitors erastin and sorafenib 314, 319 and the GPx4 inhibitor RSL3 318, 319.  Erastin, sorafenib and RSL3 induce GSH depletion and oxidative stress through the production of ROS. As a consequence, the transcription factor Nrf-2 disassociates from its inhibitory factor Keap-1 and translocates to the nucleus, where it binds to StRE/ARE sequences of target genes including HMOX1 and FTH1. HO-1 catalyzes heme degradation into CO and ferrous iron (Fe2+), both of which contribute to the pro-ferroptotic actions of HO-1 314. Fe2+ is highly reactive as a pro-oxidant and, consequently, generates ROS 322. Excessive ROS formation causes protein and lipid peroxidation leading to damage of DNA and intracellular structures, followed by the induction of ferroptosis and cell death 308, 310, 323-325. Nrf-2 induces ferritin expression to chelate Fe2+, thereby preventing the excessive generation of ROS.

 

Heme can directly activate HO-1 expression 2-4. Similar to erastin and sorafenib, BAY117089 depletes GSH and increases ROS production, leading to Nrf-2-dependent HO-1 activation and ferroptosis 313. Hassania and co-workers described the capacity of withaferin A, a steroidal lactone extracted from the roots and leaves of Withania somnifera (ashwagandha), to directly target Keap-1 and to release Nrf-2, followed by HO-1 up-regulation, iron accumulation, and cell death 312. Magnesium isoglycyrrhizinate (MgIG) was found to up-regulate HO-1 expression and free cellular iron level 317. On the other hand, the activation of HO-1 can exert a cytoprotective anti-ferroptotic effect. As demonstrated by Sun et al., ferroptosis inducers such as erastin and sorafenib are able to up-regulate ferritin expression via the Nrf-2/HO-1 pathway and thus to compensate for iron toxicity 319. Knockdown of p62, NAD(P)H:quinone oxidoreductase 1 (NQO-1), HO-1, and ferritin heavy polypeptide 1 (FTH-1) by RNA interference in vitro stimulated ferroptosis in response to erastin and sorafenib, respectively. These results led to the conclusion that the p62/Keap-1/Nrf-2 signaling pathway is crucially involved in ferroptosis via the up-regulation of down-stream anti-oxidant effectors of Nrf-2 such as HO-1, NQO-1, and ferritin 319. Data raised by Ge and colleagues clearly revealed that the Nrf-2-mediated anti-oxidant gene expression (NQO1, HMOX1, FTH1) is of avail in acquiring drug resistance 326. Artesunate, a semi-synthetic derivative of artemisin isolated from Artemisia annua, also induces the Nrf-2/HO-1 signaling cascade to support cells to acquire drug resistance 327. In this study, artesunate was shown to induce ferroptosis in head and neck cancer (HNC) cells but, however, cisplatin-resistant cells exhibited resistance to artesunate due to the activation of the Nrf2-StRE/ARE pathway, as evidenced by an up-regulation of NQO-1 and HO-1. It is of note that the inhibition of the Nrf2-StrE/ARE pathway was able to reverse ferroptosis resistance in HNC cells in vitro and in vivo 327. Likewise, withaferin A also sensitizes resistant tumor cells to existing chemotherapeutic agents 328.

 

Signaling Pathways Affected by CO

 

Heme oxygenases (HOs) catalyze the oxidative degradation of heme to equimolar amounts of biliverdin IXa, ferrous iron (Fe2+), and carbon monoxide (CO). Investigations over the past years clearly emphasize that CO exerts pleiotropic effects through a complex intracellular signaling network modulating cellular apoptotic, inflammatory, proliferative, vasoregulatory, and coagulation programs (Figure 9). The CO-dependent pathways illustrated in figure 9 are by no means exhaustive and should rather give insight into the complexity of how CO interferes with intracellular signaling cascades. Cellular targets of CO, endogeneously generated at low concentrations from heme by HOs, include the soluble guanylate cyclase (sGC) and mitogen-activated protein kinases such as p38MAPK, JNK-1/-2, and ERK-1/-2. Activation of sGC by CO leads to the formation of cyclic GMP (cGMP) culminating in anti-proliferative, anti-thrombotic, and neurotransmitter effects as well as vasodilatation 214, 329-331. Moreover, CO has also the capacity to stimulate ion channels including Ca2+-dependent K+ channels and voltage-gated L-type Ca2+ channels, contributing to vasodilatation and protection against ischemia-reperfusion (I/R) injury, respectively 332, 333. CO has been reported to modulate the activity of NO synthases (NOS) in opposing ways. On the one hand, CO inhibits the activity of NOS and down-regulates ROS production 331, 334 but, on the other hand, it stimulates NOS and consequently NO formation 335, 336. Reciprocal effects of CO can even be observed in different organgs from the same individual 335. CO has also been found to regulate cellular ROS levels by blocking complex IV (cytochrome c oxidase) of the mitochondrial respiratory chain and, thus, to up-regulate mitochondrial ROS (mtROS) formation 337-339 which can lead to autophagy 337 or cytoprotection via stabilization of HIF-1a 340. CO exerts anti-inflammatory effects through attenuation of the TLR-4-mediated cytokine production as a consequence of the blockage of NADPH oxidase-dependent ROS generation 341. Further down-stream effector molecules of CO mediating anti-inflammatory effects include Hsp70 and its transcriptional regulator HSF-1 342, peroxisome proliferator-activated receptor gamma (PPAR-g) and the pro-inflammatory transcription factor Egr-1 343, as well as Cav-1 that blocks TLR-4-dependent pro-inflammatory signal transduction 239. PPAR-g was found as being activated by CO-derived mtROS followed by the suppression of pro-inflammatory Egr-1 343. HSF-1 acts as a negative regulator of several pro-inflammatory genes, including those encoding IL-1b and TNF 344, 345. mtROS are also able to up-regulate the expression of anti-inflammatory TGF-b through stabilization of the transcription factor HIF-1a 340. It has been shown previously that TGF-b promotes activation of pro-survival genes such as HMOX1, thereby blocking inflammatory pathways 346-348. More recent investigations identified a negative regulatory role of CO in the mtROS-mediated activation of the NLRP3 inflammasome which modulates the formation of pro-inflammatory IL-1 and IL-18 349.

 

Mitogen-activated protein kinases represent crucial down-stream targets of CO. Especially activation of p38MAPK has been implicated in the anti-apoptotic, anti-inflammatory and anti-proliferative effects of CO 205, 206, 350, 351. CO exerts anti-inflammatory effects through the MKK3/p38MAPK-dependent repression of the pro-inflammatory molecules IL-1b, TNF, and macrophage inflammatory protein 1b (MIP-1b) as well as the induction of anti-inflammatory IL-10 205, 206. CO-induced activation of p38MAPK also leads to HSF-1-dependent induction of Hsp70 accompanied by a decrease in TNF and an increase in IL-10 synthesis 342. The anti-apoptotic properties of CO have been attributed to the modulation of the NF-kB signaling pathway which, upon activation, up-regulates the expression of genes coding for the anti-apoptotic proteins A1, A20, c-IAP2, and manganese superoxide dismutase (MnSOD) 350, 352. Also, Hsp70 serves as an anti-apoptotic mediator able to protect cells from cytotoxicity by blocking the caspase cascade 353-357. Further effectors of CO-mediated anti-apoptosis encompass both, the PI3K/Akt/STAT-3 and the p38MAPK/STAT-3 pathway whose activation culminates in the inhibition of pro-apoptotic molecules such as caspase-3/-8, Fas, PARP, and Bcl-2 proteins 353, 358.

 

CO has also been noted to affect the activities of ERK-1/-2 and c-Jun N-terminal kinase (JNK) pathways. In a murine model of sepsis, CO decreased the LPS-induced phosphorylation of JNK followed by the inhibition of the transcription factor AP-1 activation and consecutive down-regulation of IL-1b and IL-6 expression 359. The effects of CO on the activity of the ERK-1/-2 signaling pathway are contradictory. Basuroy et al. reported on the capacity of CO to block the activation of ERK-1/-2 in an Akt-dependent manner, a mechanism that relies on an inhibition of NOX4 NADPH oxidase and concomitant reduction of ROS production, thereby promoting cell survival during inflammatory conditions 360. In line with this notion, CO exerts anti-proliferative effects in human airway smooth muscle cell (HASMC) by attenuating ERK-1/-2 activation 215, 361. On the contrary, CO was found to inhibit extrinsic apoptosis by an activation of ERK-1/-2 leading to the blockage of caspase-8 through activation of anti-apoptotic c-FLIP (cellular Fas-associated death domain (FADD) IL-1β-converting enzyme (FLICE)-like inhibitory protein) 354. A most recent study revealed that CO activates ERK-1/-2 and confers resistance of hepatocellular carcinoma (HCC) cells to TGF-β-induced growth inhibition 362. Taking into account the results mentioned above, the signaling pathways triggered by CO can apparently diverge in different cell types and even in the same cell type in response to various cellular processes 215, 361.