Cis and trans regulation of hepcidin expression by upstream stimulatory factor

Abstract

Hepcidin is the presumed negative regulator of systemic iron levels; its expression is induced in iron overload, infection, and inflammation, and by cytokines, but is suppressed in hypoxia and anemia. Although the gene is exquisitely sensitive to changes in iron status in vivo, its mRNA is devoid of prototypical ironresponse elements, and it is therefore not obvious how it may be regulated by iron flux. The multiplicity of effectors of its expression also suggests that the transcriptional circuitry controlling the gene may be very complex indeed. In delineating enhancer elements within both the human and mouse hepcidin gene promoters, we show here that members of the basic helix-loop-helix leucine zipper (bHLH-ZIP) family of transcriptional regulators control hepcidin expression. The upstream stimulatory factor 2 (USF2), previously linked to hepcidin through gene ablation in inbred mice, appears to exert a polar or cis-acting effect, while USF1 may act in trans to control hepcidin expression. In mice, we found variation in expression of both hepcidin genes, driven by these transcription factors. In addition, c-Myc and Max synergize to control the expression of this hormone, supporting previous findings for the role of this couple in regulating iron metabolism. Transcriptional activation by both USF1/ USF2 and c-Myc/Max heterodimers occurs through E-boxes within the promoter. Site-directed mutagenesis of these elements rendered the promoter unresponsive to USF1/USF2 or c-Myc/Max. Dominant-negative mutants of USF1 and USF2 reciprocally attenuated promoter transactivation by both wild-type USF1 and USF2. Promoter occupancy by the transcription factors was confirmed by DNA-binding and chromatin immunoprecipitation assays. Taken IntroductionHepcidin is a small liver-derived acute-phase cationic peptide that is induced by iron overload, infection, and proinflammatory cytokines; on the other hand, its expression is attenuated in hypoxia and anemia. [1][2][3][4][5][6][7][8][9] The relationship between this peptide hormone and iron is analogous to the link between blood glucose levels and insulin in that iron overload is accompanied by increased hepcidin expression by the liver, while the converse is true in iron deficiency. Initially isolated from urine and by subtraction cloning of its mRNA, 1-3 this peptide has evolved as the primary regulator of iron homeostasis and a probable mediator of the anemia of chronic disease or inflammation. This role has been amply proven in a number of recent reports either by direct or indirect approaches. For example, in a study to define the role of upstream stimulatory factor 2 (USF2) in energy/glucose metabolism in mice, 10,11 hepcidin was also surprisingly identified in association with an iron overload phenotype similar to hemochromatosis, as these mice produced diminished levels of hepcidin. 12 On the other hand, transgenic mice that constitutively expressed hepcidin were severely anemic, thereby suggesting a role for this peptide in preventing iron overload. 13,14 A frame-shift ...

Association of hepcidin promoter c.-582 A>G variant and iron overload in thalassemia major

“…1,2 Hepcidin, encoded by HAMP gene, is a recently discovered 25 amino acid peptide that, in addition to being involved in innate immunity, 3 appears to play a crucial role in iron homeostasis in humans, regulating both iron absorption from the intestine and its recycling by macrophages. [4][5][6][7] It has been demonstrated that iron overload and inflammation stimulate hepcidin expression while erythropoietic activity decreases its production. Hepcidin synthesis is also suppressed by anemia and hypoxia.…”

Genetic variation in hepcidin expression and its implications for phenotypic differences in iron metabolism

“…20 However, the signals that culminate in iron-dependent hepcidin transcription or the cognate proteins that mediate this pathway remain obscure. Although we showed that USF regulates hepcidin expression, 12 the underlying signal transduction pathway remains unclear. The increasing number of transcription factors for hepcidin expression (STAT3, Smads, USF1/2, c-Myc/Max, C/EBPα, and HIF-1) has major implications because quantitative or qualitative differences in their expression (i.e.…”

“…11 Subsequently, we showed that hepcidin expression was also regulated by Upstream Stimulatory Factor (USF) and c-Myc/Max through several E-boxes with the consensus sequence CAnnTG (n is any other nucleotide); these are binding sites for the basic helixloophelix leucine zipper family of transcription factors. 12 Genes that are regulated through E-boxes including the Clock genes period, timeless and clock tend to be under circadian rhythmic transcriptional control, 13 suggesting that hepcidin may also be transcribed in pulses. This may account for the wide diurnal variations in hepcidin expression 5 which may cause cyclical changes in iron levels.…”

Serum hepcidin in infants born after 32 to 37 wk of gestational age

“…This pattern was neither influenced by food intake (11), nor by the sleep-wake cycle (26). It has been suggested that the transcriptional regulators related to genes involved in circadian rhythm (upstream stimulatory factor 1 (USF1) and USF2), are potentially significant modulators of hepcidin expression (26,27). Furthermore, diurnal variation in hepcidin might also be secondary to innate circadian rhythm of one of the many biological determinants of serum hepcidin concentrations (3,11).…”