ESTADO DE LA NORMA OHSAS 18001:2007

Keap1-Nrf2 pathway

All genes of the endogenous antioxidant response, including HO-1, have a common promoter element, the ARE, which is regulated by the Keap1-Nrf2 pathway. Keap1 (Kelch-like ECH associated protein 1) is a cysteine-rich protein that is the chemical sensor responsible for Nrf2 (nuclear factor erythroid 2-realted factor 2) activation (405). Under baseline conditions, Keap1 forms a dimer and binds cytoplasmic actin and Nrf2 and targets Nrf2 for ubiquitination, thus both physically sequestering Nrf2 in the cytoplasm and targeting it for proteolytic degradation (405). These effects of Keap1 robustly prevent Nrf2 from entering the nucleus, binding the ARE, and driving transcription of target genes. In response to numerous cellular insults, particularly oxidative stress, Keap1 releases Nrf2, allowing Nrf2 to translocate to the nucleus, heterodimerize with other transcription factors including small Maf proteins, bind the ARE, and turn on the expression of target antioxidant response genes (405). Multiple mechanisms for the release of Nrf2 by Keap1 have been demonstrated; these include Keap1 conformational changes as a result Keap1 cysteine residues changing redox state in response to an oxidizing environment (419) and ubiquitination of Keap1 (420). Additionally there is strong evidence of Keap1-independent

mechanisms of Nrf2 activation, including the ability of Nrf2 to sense inducers directly (421), increased Nrf2 mRNA transcription (422), translational control through redox-sensitive internal ribosomal entry site in the 5’-UTR of Nrf2 mRNA (423), and phosphorylation of Nrf2 (424).

The multiple pathways for activation allow the Nrf2-driven ARE to respond to a broad range of cellular insults. There is strong evidence that different cell stressors and inducers of the Nrf2- dependent ARE utilize different combinations of these mechanisms described above to ultimately induce the transcription of antioxidant response genes. Endogenously the Nrf2/ARE pathway can be upregulated by oxidative stress (e.g. reactive oxygen species, electrophiles), shear stress, endoplasmic reticulum stress, infection, inflammation, mitochondrial dysfunction, etc. (405). Additionally, exogenous compounds from up to ten distinct chemical classes have been reported to induce the Nrf2/ARE (405), highlighting the potential for therapeutic targeting of the Nrf2/ARE pathway.

Transcriptional regulation of HO-1

In addition to Nrf2, several transcription factors bind the upstream promoter region and regulate expression of the HO-1 gene including hypoxia-inducible factor-1 (425), nuclear factor-kappa B (NF-κB) (426), activator protein-1 (427), signal transducer and activator of transcription 3 (428, 429), and broad complex-tramtrack-bric-a-brac-domain (BTB) and cap’n’collar (CNC) homology 1 (BACH1) (430) (reviewed in (418)). The best studied of these regulatory transcription factors is BACH1, a potent transcriptional repressor of HO-1 in addition to some other, but not all, ARE genes (e.g. NQO1) (431). Under baseline conditions, BACH1 forms a heterodimer with small Maf proteins and can bind certain ARE promoter elements thus preventing Nrf2 binding and repressing transcription. In the presence of elevated intracellular heme levels or oxidative stress, BACH1 undergoes a conformation change and dissociates from the HO-1 promoter, allowing Nrf2 to bind and activate HO-1 gene expression (432, 433). Other pathways that have been implicated

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in regulating HO-1 gene expression include p28 MAPK (434), phosphatidylinositol-3 kinase/Akt pathway (429, 435), and the IL-10 pathway (429, 436). The multiple identified transcription factors and signaling pathways that regulate HO-1 gene expression are one reason why HO-1 protein expression is induced in response to such a wide array of cellular insults (oxidative stress, heavy metals, ultraviolet light, inflammation, etc.) and other signals.

Micro-RNA regulation of HO-1

In addition to the multiple mechanisms of transcriptional control of HO-1, there is considerable research implicating an important role for microRNA (miR) regulation of HO-1 mRNA. MicroRNAs are small non-coding RNA molecules (~22 nucleotides) that post-transcriptionally silence specific mRNAs through base-pairing with complementary sequences in the 3’ untranslated region. As a

result of this complementary binding, mRNAs can be silenced through i) mRNA cleavage and

degradation, ii) mRNA destabilization through shortening the poly(A) tail, and iii) less efficient

translation (reviewed in (437)). Multiple miRs have been identified that downregulate HO-1 expression. These HO-1-downregulating miRs include those that target HO-1 mRNA directly (e.g. miR-155 (438, 439), miR-24-3p (440), miR-377 (441), miR-217 (441), miR-122 (442), miR-16-5p (443)) and those that target other genes involved in promoting HO-1 expression (e.g. miR-34a (444)). Additionally multiple miRs have been implicated in increasing HO-1 through targeting genes involved in repressing HO-1, such as BACH1 (miR196 (445), let7 (446), and miR-155 (447)). Thus, the multifaceted transcriptional regulation of HO-1 mRNA is further nuanced through microRNA mediated regulation directly of HO-1 mRNA and indirectly through associated genes.

Posttranslational regulation of HO-1

A few studies have also examined the posttranslational regulation of HO-1. HO-1 has been shown to be ubiquitinated and subsequently degraded in a proteasome-dependent manner

through the ER-associated degradation pathway (448). The E3-ubiquitin ligase, TRC8, has been demonstrated to target HO-1 for ubiquitination and subsequent degradation (449). Whether HO-1 is regulated significantly through changes in rate of degradation is currently unknown. In addition to changes in HO-1 protein levels, HO-1 activity reportedly can be regulated through post- translational phosphorylation. Specifically, HO-1 activity can be increased, albeit modestly, through phosphorylation at serine 188 by Akt/PKB (450). In summary, HO-1 expression and activity is tightly regulated through multiple mechanisms, which is likely responsible for HO-1’s ability to be upregulated by a wide array of cellular signals and insults.