Heme Oxygenase: Regulation

Transcriptional Regulation

Heme oxygenase 1 (HO-1) represents the inducible isoform of the heme oxygenase/HSP32 family and has originally been identified as being a heat shock protein in rats ⁵³. When cells are subjected to environmental stress, they respond by increasing the expression of HSPs. The rapid induction of HSPs in response to environmental stress is based on a variety of genetic and biochemical processes referred to as the heat shock response (HSR) ²⁵³. HSR is preferentially regulated at the transcription level by heat shock factors (HSF). Among them, HSF-1 is considered as being the key transcription factor of stress-inducible HSPs ²⁵⁴. Under normal conditions, HSF-1 exists as an inactive monomer in the cytosol complexed to further chaperones such as Hsp70 and Hsp90 ²⁵⁵. In response to stress, Hsp70 and Hsp90 are released fom the complex followed by the formation of HSF-1 homotrimers capable of binding to heat shock elements (HSEs) in the promoter region of various molecular chaperone genes to induce their expression ^{256, 257}.

 

HO-1 is is encoded by a single copy gene in the human genome (HMOX1) ⁵⁵ as well as in the mouse genome (Hmox1) ⁸⁶. HO-1 is induced by a plethora of stimuli, including inflammatory mediators, chemical and physical stimuli, oxidative stress and its own substrate, heme ^2-4. The expression of HO-1 is predominantly regulated at the level of transcription ^{208, 258}. The human HMOX1 gene is approximately 14 kb long and harbors five exons and a potential HSE in the proximal promoter region ⁵⁵. Notwithstanding the above and in contrast to HO-1 from rats, human HO-1 is not induced by heat shock ⁵⁵. Continuing investigations made clear that the thermal responsiveness of HO-1 is restricted to rodents ^53-55. An interesting observation made by Sato and colleagues revealed that a novel para-hydroquinone-type pro-electrophilic compound (termed D1) induces both, the Nrf-2 and HSF-1 pathways, and may consequently provide protection against oxidative and ER stress ²⁵⁹. The promoter region of HMOX1 bears binding sites for additional transcription factors like AP-1, NF-κB, and Nrf-2 (for a review see Waza et al., 2018) ²⁶⁰. Docking analyses by Vanella and collaborators revealed a potential interaction of HO-1 with the NF-κB subunit p65/RelA ²²⁴. Several inflammatory mediators and signaling molecules such as NF-κB and TNF have been found as being strictly bound to chaperone/HSP gene expression and protein functions. In this respect, the NF-κB subunit p65/RelA acts as a transcription factor for numerous HSPs including HO-1/Hsp32 ^{224, 261-263}. The HMOX1 gene also contains functional cis-acting elements that contribute to transcriptional regulation, including the hypoxia-responsive elements identified in rodents ²⁶⁴ and a binding site for the early growth response protein 1 (Egr-1) ²⁶⁵. Amongst the numerous transcription factors, Nrf-2 is considered as being the key regulator in HMOX1 transcription ²⁶⁶. Nrf-2 binds to stress-response element/anti-oxidant response element (StRE/ARE) motifs present in the up-stream enhancer regions of the HMOX1 promoter ²⁶⁶. Under non-stress conditions, Nrf-2 is held in an inactive state in the cytoplasma in complex with the Kelch-like ECH-associated protein 1 (Keap-1) which blocks the nuclear translocation and determines the targeted ubiquitination of Nrf-2 by the cullin 3-based E3 ubiquitin ligase complex, thus identifying Nrf-2 for proteasomal degradation (Figure 6) ²⁶⁷. Stress-induced phosphorylation and activation of Keap-1 facilitates the release and translocation of Nrf-2 to the nucleus, where it forms heterodimers with small Maf proteins (cellular homologs of the v-maf oncoprotein, e.g., MafF, MafG) ²⁶⁶ followed by its binding to StRE/ARE sequences and the induction of gene transcription ²⁶⁸. The heme binding protein BTB and CNC homolog 1 (Bach-1) was recently identified as a transcriptional repressor of HMOX1. Under non-stress conditions, Bach-1 associates with small Maf proteins to form heterodimers that bind to the StRE/ARE sequences and thus competes with Nrf-2 for binding to StRE/AREs. Oxidative stress or increasing heme concentrations induce conformational re-arrangements of Bach-1 that displace Bach-1 from StRE/ARE sequences and enable its proteasomal degradation so that Nrf-2 can associate with Maf and bind to StRE/AREs within the regulatory sequences of cell defense genes ^269-271.

 

Post-transcriptional Regulation

Apart from its transcriptional regulation, HO-1 protein levels have also been found as being regulated at the post-transcriptional level. The discovery of micro-RNA (miRNA) characterized this RNA subtype as being a crucial player in regulating translation of many genes. miRNAs are a class of small non-coding RNAs that negatively regulate gene expression by binding to target mRNAs. Global alterations in miRNAs can be observed in a number of disease states including cancer ^272-274. Several investigations identified specific miRNAs that bind to the 3’-untranslated region in the HMOX1 mRNA including miR-24 ²⁷⁵, miR-29a ²⁷⁶, miR-155 ²⁷⁷, miR-200c ²⁷⁸, miR-204 ²⁷⁹, miR-211 ²⁷⁹, miR-217 ²⁸⁰, miR-377 ²⁸⁰, as well as miR-378 ²⁶. miRNAs have also been reported to affect HMOX1 expression indirectly by targeting up-stream regulators such as Bach-1 ²⁸¹, cullin 3-based E3 ubiquitin ligase ²⁸², Nrf-2 ^283-285, and Keap-1 ²⁸⁶, respectively. Alternatively, HO-1 expression was found to down-regulate the expression of certain miRNAs, including miR-1, miR-133a, miR-133b, and miR-206 ²⁸⁷. These observations underline the complexity of the post-transcriptional regulation of the HMOX1 expression by miRNAs.

 

Alternative splicing is an important post-transcriptional regulatory mechanism implicated in regulating HMOX expression. The group of Michael Bauer recently identified the (GT)_n microsatellite polymorphism as a novel regulatory mechanism in HMOX1 translation as it affects alternative splicing within the 5’-untranslated region ²⁸⁸. Microsatellite dinucleotide (GT)_n length polymorphisms in the untranslated regions of the human HMOX1 gene are known to impede the transcriptional regulation and thus to down-regulate the expression of HO-1 in persons carrying the long allele of this polymorphism ²⁸⁹. Several human diseases have been linked to HMOX1 promoter polymorphisms as summarized by Ryter and Choi ¹⁶. However, consideration should be given to negative studies demonstrating no correlation between HMOX1 polymorphisms and the outcome of certain diseases ¹⁶ thus necessitating further investigations aiming at verifying these findings.

 

Post-translational Regulation

Similar to many other chaperones, HO-1 and HO-2 are phosphoproteins ⁶⁸ in which phosphorylation regulates the activity of the molecules. Whereas HO-2 has been noted as being phosphorylated and thus activated by Ca²⁺/calmodulin ⁶⁹ and casein kinase 2 (CK-2) ⁷⁰, phosphorylation of HO-1 obviously occurs through activation of the survival kinase Akt-1 ⁷¹. Also, expression and function of HO-1 and HO-2 can be further modulated by post-translational acetylation ^{72, 73}. Acetylation of Lys243/256 in HO-1 has been implicated in HO-1-mediated tumorigenicity in xenograft models ⁷². HO turnover appears through the proteasomal system enabling efficient elimination of the molecule after ubiquitination ^{74, 75}. Only a few studies describe the palmitoylation of HO-1. In murine melanoma cells B16, palmitoylation of HO-1 was identified as a prerequisite for targeting HO-1 to the mitochondria-associated membrane (MAM), an ER-membrane domain involved in the exchange of metabolites between the ER and mitochondria ¹⁹². Palmitoylation of human HO-1 was noted in platelets even though human HO-1 lacks essential cysteinyl residues as part of a consensus motif for palmitoylation ²⁹⁰. However, further analyses are warranted in order to evaluate the relevance of HO-1 palmitoylation in vivo.

 

It is of note that the activity of HO-1 is also regulated by specific compartmentalization of the molecule. As mentioned before, a mitochondrial location of HO-1 was found as being associated with a marked increase in the heme oxygenase activity in lung epithelial cells exposed to cigarette smoke extract or hemin ⁸, while its localization at the plasma membrane reduces its catalytic activity through an interaction with Cav-1 acting as a competitive inhibitor of HO-1 ^{9, 194}. Data raised by Weng et al. convincingly demonstrated that Cav-1 scaffolding domain (CSD) peptides reduced the compartmentalization of HO-1 and Cav-1, and increased the HO-1 activity both in lipopolysaccharide (LPS)-treated alveolar macrophages and in mice ²⁹¹.