Heme Oxygenase: 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.
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.
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.