The nuclear repressor BACH is known to
The nuclear repressor BACH1 is known to control HO-1 expression together with the transcriptional activator NRF2. The results of our study show that HO-1 gene expression in LPS-stimulated macrophages is primarily regulated via BACH1. This idea is supported by a series of experimental evidence demonstrating that: 1) even though NRF2 is activated by LPS in both human and mouse cells, HO-1 is decreased in correlation with BACH1 accumulation in hMDMs but is induced in association with BACH1 disappearance in mBMDMs; 2) in NRF2−/− mBMDMs, LPS still elicits up-regulation of HO-1 together with decreases of BACH1 and 3) NQO1, a prototypical gene regulated by NRF2, is equally augmented by LPS in human and mouse cells. Notably, our current findings are in line with previous reports in which knockdown of BACH1 in human keratinocytes was specific for HO-1 regulation, whereas knockdown of NRF2 was associated with regulation of numerous inducible antioxidant and detoxification genes [45,46]. A central issue in our study is the significance of changes in labile heme levels in the macrophage response to inflammatory stimuli. In the current study endogenous labile heme was quantified using an enzymatic apo-HRP assay previously described [21,26], which was specifically developed for detection of labile (or regulatory) heme [11,25,26]. In LDC000067 to exchange-inert heme, which is primarily allocated to hemoproteins, regulatory heme is only loosely bound to non-hemoproteins and can be readily mobilized for heme-dependent signaling and synthesis of hemoproteins [22,47]. Our data demonstrate that levels of labile heme are modulated in macrophages following LPS activation, suggesting a mobilization of loosely-bound heme that subsequently acts as a signal to regulate BACH1 and HO-1 expression. In addition, levels of labile heme change also in unstimulated macrophages lacking either BACH1 or HO-1. It is intriguing to observe that in the absence of BACH1, mouse and human (in which BACH1 was silenced by siRNA) macrophages exhibit an increase in labile heme under basal conditions. This effect is evident despite induction of HO-1, which would be expected to degrade all excess heme available. Accordingly, silencing of HO-1 in human cells is accompanied by a sharp decrease in labile heme with marked overabundance of BACH1 protein. Thus, we are tempted to postulate that the labile heme pool is not accessible to HO for degradation, but serves as unique intracellular signal to regulate BACH1 expression. Whether this is a consequence of a higher affinity for or better access of BACH1 to labile heme compared to other heme binding proteins remains an open question. Concerning the origin of labile heme, our data using the inhibitor of ALAS SA indicate that newly synthesized heme substantially contributes to the maintenance of this regulatory heme pool. It is important to note that regulation of intracellular labile heme levels is not only governed by heme-degrading HOs and the heme-synthesizing enzyme ALAS, but also by various heme-binding proteins such as glutathione-S-transferases, heme-binding protein 23 and GAPDH [24,, , , ]. Based on our current findings it is conceivable that upon inflammatory activation of macrophages, rapidly available heme is mobilized from the intracellular labile heme pool to provide the prosthetic group for the synthesis of various inducible hemoproteins including COX-2, iNOS, and NADPH oxidase-2 (NOX2) [43,51,52]. A minor fraction of so-called ‘free’ heme may also exist even under physiological conditions as discussed by various authors [11,47,49]. However, because ‘free’ heme can be cytotoxic via the generation excess reactive oxygen species (ROS), this issue is controversially discussed. For example, in macrophages high concentrations of heme have been shown to cause necrotic cell death and heme has recently also been proposed to be an alarmin [28,53]. Our finding that HO-1 regulation by exogenous heme was not blocked by the pharmacological TLR4 inhibitor TAK-242 in hMDMs suggests that TLR4-independent pathways may also be involved in mediating heme-dependent effects in macrophages, which adds to the on-going discussion on the potential mechanisms of heme signaling [27,54,55].