Regulation of heme oxygenase-1 gene expression through the phosphatidylinositol 3-kinase/PKC-ζ pathway and Sp1

Abstract

The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1–Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-ζ expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.

Introduction

Neurons, on one hand, exhibit high oxygen consumption and produce oxidizable substances such as unsaturated lipids and catecholamines and, on the other, express low levels of antioxidant enzymes [1]. This precarious balance makes them particularly sensitive to oxidative damage. NGF exerts pleiotropic actions on target neurons, such as cessation of cell division or differentiation to a neuronal phenotype but also induction of tolerance to oxidative stress, and promotion of cell survival [2]. A prominent mechanism involved in NGF-induced cell survival includes the activation of the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide dependent kinase-1 (PDK-1) axis and its downstream effectors, the Ser/Thr protein kinases Akt/PKB and atypical protein kinase C zeta (PKC-ζ) [3], [4]. Although PI3K exerts protective actions against oxidative damage in central and peripheral neurons [5], the mechanism whereby this pathway prevents reactive-oxygen-species-induced neuronal death still remains poorly defined. In this context, we have reported the upregulation of HO-1 expression by NGF in a PI3K-dependent manner and its relevance in cytoprotection against the parkinsonian neurotoxin 6-hydroxidopamine [6]. Consistent with our observations, other groups have found that inhibitors of PI3K block the induction of HO-1 expression in other cellular contexts including the response to IL-10, hepatocyte growth factor [7], [8], endotoxin, arsenite, hemin, and carnosol [9], [10], [11], [12]. However, the pathways involved in this regulation remain unexplored.

Emerging evidence supports a role for moderate upregulation of heme oxygenase-1 in antioxidant protection and survival of nerve-derived cells [13], [14]. The heme oxygenase family is composed of at least two well-characterized isoenzymes: inducible HO-1 and constitutive HO-2. Both HO isoforms are different gene products and dissimilar in their tissue distribution and mode of regulation. Nevertheless, they catalyze the degradation of heme to release free iron, carbon monoxide, and the linear tetrapyrrole biliverdin, the later being converted to bilirubin by the enzyme biliverdin reductase. While HO-2 may have a principal role in the release of the gas neurotransmitter carbon monoxide, HO-1 appears to be more involved in protection against oxidative stress [15]. The normally low HO-1 protein levels in neurons [16], [17], increase after cerebral ischemia [18], [19], in the formation of brain neurofibrillary tangles in Alzheimer's patients [20] and in neuronal Lewy bodies of Parkinson's disease patients [21]. Heme and other stress stimuli that produce oxidative stress (UV light, heavy metals, glutathione depletion, H₂O₂) enhance HO-1 expression in practically all tissues and cells tested including neurons [22]. Therefore, induction of a moderate intracellular heme catabolism through HO-1 represents an adaptive and ultimately protective response to oxidative injury.

HO-1 is subjected to complex transcriptional regulation. A large variety of putative regulatory elements in the 5′ region of the mouse, rat, chicken, and human ho1 promoters has been described [23], [24], [25]. These elements account for the transcriptional responses to oxidant and nonoxidant stimuli. While electrophiles appear to target antioxidant response elements (AREs), NF-κB sites, or heat shock response elements (HREs), little is known about its regulation by nonoxidant stimuli, such as NGF. Moreover, the mechanistic connection between the general survival pathway represented by PI3K and the antioxidant pathway that involves HO-1 has not been established.

In this study, we have analyzed the regulation by NGF and PI3K on the 4.0-kb upstream regulatory sequence of the human ho1 promoter. We have identified a new Sp1 regulatory element that participates in the NGF- and PI3K-induced upregulation of HO-1 expression. Moreover, we demonstrate roles of PKC-ζ and the MAP kinase cascade MEK/ERK, but not Akt1, in the PI3K-induced regulation of HO-1 expression by modulating Sp1 activity.