Evidence for a lack of a direct transcriptional suppression of the iron regulatory peptide hepcidin by hypoxia-inducible factors.

BACKGROUND: Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation. METHODOLOGY/PRINCIPAL FINDINGS: Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1alpha or HIF-2alpha knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased. CONCLUSIONS/SIGNIFICANCE: Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression

Cytostatic potential of novel agents that inhibit the regulation of intracellular pH

Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH (pHi) through the activity of membrane-based ion exchange mechanisms including the Na+/H+ antiport and the Na+-dependent Cl−/HCO3− exchanger. Inhibition of these regulatory mechanisms has been proposed as an approach to tumour therapy. Previously available inhibitors of these exchangers were toxic (e.g. 4,4-diisothiocyanstilbene-2,2-disulphonic acid), and/or non-specific (e.g. 5-N-ethyl-N-isopropyl amiloride). Using two human (MCF7, MDA-MB231) and one murine (EMT6) breast cancer cell lines, we evaluated the influence of two new agents, cariporide (an inhibitor of the Na+/H+ antiport) and S3705 (an inhibitor of the Na+-dependent Cl−/HCO3− exchanger) on the regulation of intracellular pH (pHi). The cytotoxicity of the two agents was assessed by using clonogenic assays. Our results suggest that cariporide has similar efficacy and potency to 5-N-ethyl-N-isopropyl amiloride for inhibition of Na+/H+ exchange while S3705 is more potent and efficient than 4,4-diisothiocyanstilbene-2,2-disulphonic acid in inhibiting Na+-dependent Cl−/HCO3− exchange. The agents inhibited the growth of tumour cells when they were incubated at low pHe (7.0–6.8), but were non-toxic to cells grown at doses that inhibited the regulation of pHi. Our results indicate that cariporide and S3705 are selective cytostatic agents under in vitro conditions that reflect the slightly acidic microenvironment found in solid tumours

(Pro)renin receptor peptide inhibitor "handle-region" peptide does not affect hypertensive nephrosclerosis in goldblatt rats

The (pro)renin receptor [(P)RR], a new component the renin-angiotensin system, was cloned recently. The (P)RR promotes direct mitogen-activated protein kinase signaling and nonproteolytic prorenin activation. We investigated the role of a (P)RR blocker, a peptide consisting of 10 amino acids from the prorenin prosegment called the "handle-region" peptide (HRP), on target organ damage in renovascular hypertensive 2-kidney, 1-clip (2K1C) rats. Vehicle-treated 2K1C rats were compared with HRP-treated 2K1C rats (3.5 microg/kg per day) and sham-operated controls. Vehicle-treated 2K1C rats developed hypertension (186+/-17 mm Hg), cardiac hypertrophy (3.16+/-0.16 mg/g), renal inflammation, fibrosis, vascular, and tubular damage. Chronic HRP treatment did not affect blood pressure (194+/-15 mm Hg), cardiac hypertrophy (2.97+/-0.11 mg/g), or renal damage. Furthermore, we investigated the renal renin and (P)RR expression. The clipped kidney of 2K1C and HRP-treated 2K1C rats showed a higher renin expression and juxtaglomerular index compared with sham-operated kidneys. The unclipped kidney showed suppressed renin expression. In contrast, (P)RR mRNA expression was not altered in any group. Plasma renin activity and aldosterone were increased in 2K1C rats compared with sham controls. HRP-treated 2K1C rats tended to lower plasma renin activity but showed similar aldosterone levels as vehicle-treated 2K1C rats. Our results indicate that blockade of the (P)RR with HRP does not improve target organ damage in renovascular hypertensive rats