Heme is an essential molecule with contradictory biological functions. In hemoproteins such as hemoglobin and cytochromes protein-bound heme is a prosthetic group serving physiological functions as a transporter for oxygen and electrons. On the other hand free heme can have deleterious effects by generating reactive oxygen species that cause oxidative stress. Consequently, heme homeostasis of the cell must be tightly controlled. The biosynthesis of heme is catalyzed by eight enzymes that are differentially regulated in liver and erythroid cells. Recent findings on proinflammatory functions of heme and its role in the pathogenesis of diseases, such as rhabdomyolysis or atherosclerosis are summarized. The regulation of gene expression by heme in yeast and mammalian cells and the underlying molecular mechanisms are presented. Finally, we discuss the functional significance of the heme-degrading enzyme heme oxygenase and hemebinding proteins for the regulation of heme homeostasis.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and modulates the inflammatory immune response. Because HO-1 is up-regulated by NAD(P)H oxidase activators such as lipopolysaccharide and 12-O-tetradecanoylphorbol-13-acetate in monocytic cells, we investigated the gene regulation of HO-1 by the chemical NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Unexpectedly, AEBSF induced endogenous gene expression and promoter activity of HO-1 in cell cultures of human and mouse monocytes. Inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PKB) pathway by pharmacological inhibitors and cotransfection of an expression vector for a dominant negative mutant of PKB reduced the AEBSF-dependent induction of HO-1 gene transcription. Accordingly, overexpressed constitutively active PKB markedly up-regulated HO-1 promoter activity. AEBSF activated the mitogen-activated protein kinases (MAPK) JNK and p38. Inhibition of p38α and p38β, but not that of JNK or p38γ and p38δ, prevented the induction of HO-1 gene expression by AEBSF. p38 was stimulated by AEBSF in a PKB-dependent manner as demonstrated by a luciferase assay with a Gal4-CHOP fusion protein. Finally, AEBSF- and PKB-dependent induction of HO-1 promoter activity was reduced by simultaneous mutation of an E-box motif (–47/–42) and a cAMP response element/AP-1 element (–664/–657) of the proximal HO-1 gene promoter. Overexpression of the basic helix-loop-helix transcription factor USF2 and coactivator p300 enhanced the AEBSF-dependent response of the HO-1 promoter. The data suggest that the transcriptional induction of HO-1 gene expression by AEBSF is mediated via activation of a PKB, p38 MAPK signaling pathway.

The antioxidant protein peroxiredoxin (Prx) I is a thioredoxin peroxidase that is involved in the regulation of proliferation and differentiation of mammalian cells. Here, it is shown that Prx I gene expression was induced transcriptionally by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cultured rat liver tissue macrophages and RAW264.7 monocytic cells. TPA-dependent induction of Prx I gene expression was mediated by two proximal activator protein-1 sites of the rat Prx I promoter region that were nuclear targets of c-Jun as determined by transfection studies with luciferase reporter gene constructs and electrophoretic mobility shift assays. The transcription factor Nrf2, however, was not involved in the regulation of Prx I promoter activity. Prx I gene induction by TPA was decreased by protein kinase C inhibitors and overexpressed dominant negative forms of Ras and MEKK1, but not Raf-1. The p38 MAPK inhibitor SB202190 and overexpression of dominant negative mutants of MAPK kinase 4 (MKK4), MKK6, and p38 inhibited the TPA-dependent induction of Prx I gene transcription. In contrast, inhibitors of the JNK, SP600125, and the NF-κB signaling pathway, caffeic acid phenethyl ester, respectively, as well as overexpressed dominant negative MKK7 and IκB, had no effect on the up-regulation of Prx I reporter gene activity by TPA. Cotransfection of wild-type p38α and p38β, but not that of p38γ and p38δ, increased Prx I promoter activity. The data indicate that a protein kinase C, Ras, MEKK1, p38 MAPK signaling pathway plays a major role for the transcriptional up-regulation of Prx I gene expression.

SUMMARY: Orangutans (Pongospp.), Asia's only great apes, are threatened in their survival due to habitat loss, hunting and infections. Nematodes of the genusStrongyloidesmay represent a severe cause of death in wild and captive individuals. In order to better understand whichStrongyloidesspecies/subspecies infect orangutans under different conditions, larvae were isolated from fecal material collected in Indonesia from 9 captive, 2 semi-captive and 9 wild individuals, 18 captive groups of Bornean orangutans and from 1 human working with wild orangutans. Genotyping was done at the genomic rDNA locus (part of the 18S rRNA gene and internal transcribed spacer 1, ITS1) by sequencing amplicons. Thirty isolates, including the one from the human, could be identified asS. fuelleborni fuelleborniwith 18S rRNA gene identities of 98·5–100%, with a corresponding published sequence. The ITS1 sequences could be determined for 17 of these isolates revealing a huge variability and 2 main clusters without obvious pattern with regard to attributes of the hosts. The ITS1 amplicons of 2 isolates were cloned and sequenced, revealing considerable variability indicative of mixed infections. One isolate from a captive individual was identified asS. stercoralis(18S rRNA) and showed 99% identity (ITS1) withS. stercoralissequences from geographically distinct locations and host species. The findings are significant with regard to the zoonotic nature of these parasites and might contribute to the conservation of remaining orangutan populations.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation, which is up-regulated by a host of stress stimuli. The bacterial cell membrane component lipopolysaccharide (LPS) is a prototypical activator of monocytic cells. Here, it is shown that LPS induced the endogenous HO-1 gene expression in RAW264.7 monocytic cells. To investigate the molecular mechanisms of HO-1 gene induction by LPS, we performed transfection experiments with reporter gene constructs containing sequences of the proximal rat HO-1 gene promoter. Deletion and mutation analysis indicated that a cyclic AMP response element/activator protein-1 site (–664/–657), but not an E-box motif (–47/–42), played a major role for LPS-dependent HO-1 gene induction. Up-regulation of HO-1 promoter activity by LPS was decreased by pharmacological nuclear factor-kB (NF-kB) inhibitors and by cotransfected expression vectors with dominant negative isoforms of NF-kB-inducing kinase, inhibitor of NF-kB (IkB) kinase β, and IkBα. Moreover, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and overexpressed dominant negative p38β decreased, whereas dominant negative p38δ increased, LPS-dependent induction of HO-1 gene expression. The results suggest that the NF-kB and p38 MAPK signaling pathways mediate the LPS-dependent induction of HO-1 gene expression via DNA sequences of the proximal promoter region. Antioxid. Redox Signal. 6, 802–810.