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 IXα, 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 highlighting 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 (T_reg) 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 research 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-κB ^229-231. In line with these findings, docking analyses by Vanella and collaborators revealed a potential interaction of HO-1 with the NF-κB 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 resistance 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 hemin⁸. 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.