Heme oxygenase (HO) controls the rate limiting reaction in the degradation of heme to biliverdin. Biliverdin is then rapidly converted to bilirubin by biliverdin reductase. The enzyme exists in two isofoms: HO-1 and HO-2. HO-1 is highly inducible by conditions such as oxidative stress [1
], or by agents like heme [2
], UVA [4
], lipopolysaccharide (LPS) [5
], interleukin-1 (IL-1) [6
], and tumor necrosis factor-alpha (TNF-α) [6
] in vivo
and in vitro
. HO-2 is a housekeeping enzyme expressed constitutively in almost all tissues.
The induction of HO-1 by various stressors is mainly regulated at the transcriptional level. Several transcriptional activators, such as Nrf2 and NFκB, bind to the consensus sequences in the HO-1 promoter and enhancers located 15 kb upstream from the HO-1 coding sequence and upregulate HO-1 transcription [7
]. Recently, Bach1, a leucine zipper protein, has been identified as a repressor of the HO-1 gene [8
]. It binds to the MARE (Maf recognition element) regions within distal enhancers-1 and -2 (DE1 and DE2, respectively) of the HO-1 promoter by heterodimerizing to MafG or MafK proteins in the absence of heme. Heme is not only the substrate for HO enzyme, but also the natural inducer of HO-1 expression. Upon exposure to heme, Bach1 dissociates from its heterodimerization partners and is exported out of the nucleus by a Crm1-dependent mechanism [9
]. Displacement of Bach1 leads to recruitment of Nrf2, also a leucine zipper protein that binds to HO-1 promoter by cooperating with MafG and MafK proteins, to activate gene expression [10
]. In addition, heme can stabilize the Nrf2 protein and lead to the accumulation of heterodimers of Nrf2/MafG that bind to MARE to activate HO-1 transcription [11
]. These mechanisms all lead to a higher expression of HO-1. In contrast, the induction of HO-1 transcription by cadmium chloride, a strong inducer of HO-1 with no structural similarities to heme, also involves the nuclear export of Bach1 via an ERK1/2 dependent pathway to activate HO-1 gene expression [12
]. Therefore, the expression of HO-1 by different inducers involves both activation of positive inducers, such as Nrf2, and inactivation of gene repressors, like Bach1, to allow for gene transcription.
In our studies investigating the transcriptional regulation of HO-1, we have found that some heme analogs, metalloporphyrins, are potent competitive inhibitors of HO activity both in vitro
and in vivo
. However, although metalloporphyrins has been shown to suppress HO activity, some metalloporphyrins [e.g.,
tin mesoporphyrin (SnMP)] can also induce its transcription via direct effects on the HO-1 promoter [3
To understand the mechanism by which SnMP induces the expression of HO-1 with a simultaneous inhibition of HO activity [2
], we investigated the effects of SnMP on HO-1, HO-2, and Bach1 mRNA, protein, and protein stability. We also used shRNA to study the direct involvement of Bach-1 in HO-1 regulation. We hypothesized that SnMP binds to the heme-binding region of Bach1 and causes it to detach from the DNA-binding complex relieving the repression of the MARE site within the HO-1 promoter, and thus activating HO-1 gene expression.