Heme Oxygenase: Function

Heme oxygenases (HOs) exhibit distinct functions at diverse cellular compartments. HOs are ER-associated oxygen oxidoreductases involved in the physiological disintegration of heme into equimolar amounts of biliverdin IXa, CO, and Fe2+ (Figure 4) 1. Heme represents an iron-containing tetrapyrrole complex that exerts potential pro-oxidant and pro-inflammatory activities. In mammals, two main isoforms are expressed: HO-1 and HO-2. While HO-2 is constitutively expressed under basic conditions in the majority of human tissues, HO-1 represents the inducible HO variant whose expression is highly up-regulated upon exposure to different kinds of stress 2-4. HOs exert cytoprotective effects by generating anti-oxidant and anti-inflammatory mediators, such as CO and biliverdin, the latter being converted to bilirubin by biliverdin reductase. Biliverdin and bilirubin are able to scavenge reactive oxygen species (ROS) 198, 199 and thereby to block lipid and protein peroxidation 200, 201. Moreover, bilirubin has been characterized to exert strong anti-apoptotic 202 and anti-inflammatory effects 203.

 

The cytoprotective capacity of HOs is also facilitated by a further degradation product, CO which functions as a crucial second messenger implicated in several signal transduction pathways, such as production of anti-inflammatory cytokines, up-regulation of anti-apoptotic effectors, and thrombosis. CO acts as a potent anti-oxidant 204, 205 and exhibits anti-inflammatory and anti-apoptotic properties via modulation of the mitogen-activated protein kinase pathway (MAPK), activation of soluble guanylyl cyclase (sGC), and up-regulation of cGMP 202, 206-209. CO-induced up-regulation of cGMP has been demonstrated to modulate vascular tone 207, 210, thereby hightlighting the crucial and well-known function of CO in vasoregulation. HO-derived CO also participates in blood vessel formation 211, induction of VEGF generation 212, and proliferation of endothelial cells (ECs) 213. Pleiotropic effects of HO-derived CO also include blockage of smooth muscle cell (SMC) proliferation 214, 215 and platelet aggregation 216, as well as stimulation of fibrinolysis 217 and inhibition of matrix deposition 218. HO-1/CO has also been found to exert immunomodulatory effects by regulating the functions of antigen-presenting cells (APCs), dendritic cells (DCs), and regulatory T (Treg) cells (for a review see Ryter and Choi, 2016) 16. Activation of HO-1 also up-regulates the Fe2+-mediated ferritin expression which competitively inhibits Fe2+, thus detoxifying its pro-oxidant activity 219, 220.

 

Recent evidence suggests that HO-1 possesses additional physiological functions apart from its enzymatic activity, termed non-canonical functions (Figure 5) 17. In this context, HO isoforms have recently been identified to perform protein-protein interactions and thus to act as molecular chaperones. The group of Phyllis A. Dennery convincingly revealed an interaction between the N-terminal regions of HO-1 and HO-2 which leads to a decrease in the total enzymatic activity 221. Form these findings the authors speculated that this HO-1/HO-2 interaction may function in limiting the HO enzymatic activity and thus promoting the non-canonical functions of HOs, particularly of HO-1.  A potential immunoregulatory function of HO-1 was proposed by Li Volti and co-workers who determined HO-1 in human milk 196. Computational analyses by the same group identified the oxidized LDL-binding protein CD91/Lrp-1 as a potential extracellular interactor of HO-1.  As CD91 is localized on various cell types including APCs 222, 223 where it serves as the common receptor for all immunogenic HSPs 222, a putative role of HO-1 as a chaperokine in immune response regulation became apparent.

 

Several investigations also shed light on the interaction of HO-2 with other proteins.  Vanella et al. identified adiponectin as a novel interactor of HO-2 and described HO-2 as being a chaperone for the physiological secretion of adiponectin from the ER 224. Adiponectin, which is synthesized in the ER, belongs to the class of adipokynes and requires specific chaperone activity for its maturation and secretion into the extracellular space 225. Thus, adiponection/HO-2 interactions highlight the role of HO-2 as a molecular chaperone for the adiponectin assembly 224. Fluorescence resonance energy transfer (FRET) experiments revealed an interaction between HO-2 and cytochrome p450 reductase (CPR) in the generation of a dynamic transitional protein complex preceding the assembly of the electron transfer complex 226. Moreover, HO-2 was noted as being part of the Ca2+-sensitive potassium (BK) channel complex enhancing channel activity under normoxic conditions 227. Particularly, inhibition of BK channels by hypoxia has been found to rely on the presence of HO-2 and this inhibition could be intensified by HO-2 stimulation. Based on these observations one can propose that HO-2 serves as an oxygen sensor controlling channel activity during oxygen deprivation 227.

 

HO-1 and HO-2 have originally been characterized as being anchored with membranes of the smooth ER via their C-terminal TMS 67. Intriguing reseach clearly revealed an association of HO-1 with extra-ER compartments, including the cytosol 5, 6, mitochondria 7, 8, plasma membrane caveolae 9, chloroplasts 10-13, extracellular space 14, and the nucleus 15. In the ER, caveolae and mitochondria, TMS-anchored HO-1 appears to co-localize with cytochrome p450 reductase (CPR) and biliverdin reductase (BVR), implying heme degradation as being its primary function 7, 21. Nuclear HO-1 was found as being catalytically inactive, but it might function as a transcriptional regulator 15, 191 and a modulator of cytoprotective mechanisms 228, respectively. With respect to transcriptional regulation, HO-1 translocates to the nucleus under hypoxic conditions and enhances the activation of anti-oxidant responsive promoters and transcription factors such as AP-1 and NF-kB 229-231. In line with these findings, docking analyses by Vanella and collaborators revealed a potential interaction of HO-1 with the NF-kB subunit p65/RelA 224 which might contribute to anti-apoptotic functions. It is interesting to note that the nuclear location of HO-1 is accompanied by a dramatic reduction in enzymatic activity 232, confers resistence of certain tumor cells to chemotherapy 233, 234, and has been linked to genetic instability 234. Nuclear translocation is facilitated by the signal peptide peptidase (SPP)-mediated intra-membrane cleavage of  HO-1, leading to the formation of a truncated HO-1 variant devoid of the TMS 235. In this case, nuclear HO-1 translocation was reported to promote cancer cell proliferation and invasion suggesting a pivotal role of HO-1 in cancer progression independent of its enzymatic activity.

 

HO-1 can also locate to mitochondria where it associates with the mitochondrial inner membrane even though it lacks a regular mitochondrial targeting sequence 7. It has been suggested that mitochondrial-associated membranes (MAM) might be implicated in the trafficking of HO-1 between mitochondria and the ER 21, 192. Work by Converso and collaborators disclose an important role of HO-1 in mitochondrial heme turnover and a protective function in pathophysiological situations, including sepsis, neurodegeneration and hypoxia 7. Mitochondrial location of HO-1 was also associated with a marked increase in the heme oxygenase activity in lung epithelial cells exposed to cigarette smoke extract or hemin 8. Further pathological conditions such as gastric mucosal injury also induce the mitochondrial translocation of HO-1 followed by the activation of cytoprotective and anti-apoptotic pathways 236. In this study, free heme was noted to accumulate in mitochondria during gastric injury and mitochondrial entry of HO-1 decreased intra-mitochondrial free heme, underlining the importance of mitochondrial HO-1 translocation in the detoxification of aggregated heme 236. However, continuing mitochondrial translocation of HO-1 may certainly have negative effects on cell physiology because it has been shown to restrict the synthesis of heme-containing mitochondrial proteins 237 and to up-regulate ROS production and to mediate mitochondrial dysfunction as evidenced by an increase in autophagy and cytochrome c release 238.

 

HO-1 may be shipped to caveolae and the plasma membrane via the ER and Golgi apparatus where it also contributes to heme detoxification. Herein, HO-1 interacts with the caveola-specific membrane proteins caveolin 1 (Cav-1) and Cav-2, respectively 9, 193, 194. The interaction of HO-1 with Cav-1 was noted to reduce the activity of HO-1, indicative for a negative regulatory role of Cav-1 9. HO-1 translocation to caveolae obviously occurs in a p38MAPK-dependent manner, leading to the interaction of Cav-1 with Toll-like receptor 4 (TLR-4) and the down-regulation of pro-inflammatory signaling pathways 239. A growing body of evidence now indicates that Cav-1 acts a competitive inhibitor of HO-1 194.

 

As already stated, HO-1 can be found in various extracellular compartments including body fluids. HO-1 was identified not only in human milk 196 but also the serum and plasma as well as the cerebrospinal fluid of healthy and diseased human individuals (summarized by Vanella et al., 2016) 17. Extracellularly located HO-1 might function as a potential biomarker in disease 18, 19 or as an extracellular receptor ligand 20. Compared to controls, enhanced serum/plasma levels of HO-1 were determined, e.g., in patients with pre-eclampsia 14, Alzheimer’s disease (AD) 240, Parkinson’s disease (PD) 241, in patients resuscitated from out-of-hospital cardiac arrest 242, acute kidney injury 243, interstitial pneumonia 244, silicosis 245, 246, in patients with acute respiratory distress syndrome (ARDS) 247, 248, and adult-onset Still’s disease (AOSD) 249. Decreased serum/plasma levels are observed in patients with type 2 diabetes mellitus (T2DM) 250, gestational diabetes mellitus 251, and laryngeal squamous cell carcinoma 252.