Hepcidin is a peptide that regulates iron homeostasis by inhibiting iron absorption by the small intestine and release of iron from macrophages. Its production is stimulated by iron overload and by inflammation. It has been suggested that IL-6 is the only cytokine that stimulates hepcidin transcription. However, mice with targeted disruption of the gene encoding IL-6 (IL-6 ؊/؊ ) respond to endotoxin by increasing the expression of hepcidin transcripts in the liver. We show that incubating murine hepatocytes with IL-6, IL-1␣, and IL-1␤ strongly stimulates hepcidin transcription. IL-10 has little or no stimulatory effect, and IFN-␤ inhibits transcription of hepcidin. All of the hepcidin stimulatory activity of macrophages from IL-6 ؊/؊ mice can be accounted for by IL-1 that they secrete. Hepatocytes from IL-6 ؊/؊ mice, hfe ؊/؊ mice, and mice with a hypomorphic transferrin receptor 2 mutation responded to IL-6 and IL-1 by up-regulating hepcidin transcription. Nitric oxide does not seem to be involved in the stimulation of hepcidin transcription by cytokines: aminoguanidine does not inhibit the stimulation of hepcidin transcription by cytokines. IL-1 may play a significant role in the anemia of inflammation by up-regulating hepcidin.HFE ͉ iron ͉ liver ͉ nitric oxide H epcidin has emerged as a central regulator of iron homeostasis. First described as a 25-amino acid antimicrobial peptide (1, 2), it was subsequently found to be a powerful negative regulator of iron absorption (3, 4). Befitting its role as an antimicrobial peptide, hepcidin is up-regulated in intact animals by the injection of endotoxin or turpentine.Moreover, culture media conditioned by treatment of macrophages with LPS stimulate hepcidin transcription in cultures of primary hepatocytes (5). Because this stimulation was entirely blocked by anti-IL-6 antibody, it was concluded that the stimulation was due to IL-6 and that stimulation did not occur with IL-1 and TNF-␣ (6). Our data suggested, however, that some stimulation of hepcidin production occurred in IL-6 Ϫ/Ϫ mice treated with LPS (7). These data have recently been confirmed by Rivera et al.† Accordingly, there must be substances other than IL-6 that stimulate hepcidin production. We now show that hepatocytes can be stimulated directly to produce hepcidin message by the cytokines IL-6, IL-1␣, and IL-1␤ and that the stimulation of hepcidin production by macrophage-conditioned media can be accounted for entirely by these three cytokines. Materials and MethodsAll cytokines and cytokine-specific antibodies were obtained from R & D Systems. Aminoguanidine hemisulfate was purchased from Calbiochem. IL-6 Ϫ/Ϫ mice were on a background of C57BL͞6J (002650) and were obtained from The Jackson Laboratory. hfe Ϫ/Ϫ mice and transferrin receptor 2 (tfr2) mutant mice were kind gifts from Dr. William Sly (Saint Louis University, St. Louis) and Dr. Robert Fleming (Saint Louis University), respectively. The hfe Ϫ/Ϫ mice had been backcrossed into the 129 strain for 10 generations; the tfr2 mutant mice, transgenic m...

Recently, it has been suggested that hepcidin, a peptide involved in iron homeostasis, is regulated by bone morphogenetic proteins (BMPs), apparently by binding to hemojuvelin (Hjv) as a coreceptor and signaling through Smad4. We investigate the role of Hfe, Tfr2 (transferrin receptor 2), and IL-6 in BMP2-, BMP4-, and BMP9-stimulated up-regulation of murine hepcidin, because these molecules, like Hjv, are known to be involved in hepcidin signaling. We show that the BMP signaling pathway acts independently of Hfe, Tfr2, and IL-6: The response to BMP2, BMP4, and BMP9 is similar in isolated hepatocytes of wild-type, Hfe ؊/؊ , IL-6 ؊/؊ , and Tfr2 m mutant mice. The potency of different human BMPs in stimulating hepcidin transcription by murine primary hepatocytes is BMP9 > BMP4 > BMP2. However, in human HepG2 cells, BMP4 and BMP9 are equally potent, whereas BMP2 requires a higher dose to become an effective hepcidin activator. Moreover, all of the tested BMPs are more potent regulators of hepcidin than IL-6 and thus are the most potent known stimulators of hepcidin transcription.one morphogenetic proteins (BMPs) are cytokines belonging to the TGF-␤ superfamily. They play a crucial role in regulating cell proliferation, cell differentiation, and apoptosis and in the development of tissues (1, 2). There are 20 different human BMPs, with various expression profiles and tissue distribution. BMPs function by binding to specific receptors, which are divided into two separate groups: BMP receptor type I and type II. Binding to receptor homodimers is very weak, and highaffinity binding is accomplished by forming heterodimers of type I͞type II receptors (3,4).Formation of the BMP-BMP receptor I͞II type complex places these receptors in close proximity, leading to phosphorylation of the BMP type I subunit by the constitutively active serine͞threonine kinase type II receptor. Phosphorylated receptor I is an active kinase that subsequently phosphorylates intracellular messengers of BMP signaling, including the Smad proteins and mitogen-activated protein kinase (5).Smad proteins can be divided into three groups: receptormediated Smads (R-Smads) (Smad1, -2, -3, -5, and -8), the common mediator Smads (Co-Smads) (Smad4), and inhibitory Smads (Smad6 and -7). R-Smads are associated with the receptor complex and, upon ligand binding, become phosphorylated and form heterodimers with Co-Smad. The R-Smad͞Co-Smad complexes translocate to the nucleus, where they bind directly or through specific transcriptional partners to promoter sequences of the target, regulated genes that are responsible for the transcriptional response to BMPs (6).Recently, BMPs have been found to have a previously unexpected role in iron metabolism. Babitt et al. (7) demonstrated that RGMa, a homolog closely related to the protein associated with juvenile hemochromatosis (hemojuvelin, Hjv), was a coreceptor for BMP2 and BMP4. Babitt et al. (8) subsequently showed that Hjv was also a coreceptor for BMPs and suggested that the Hjv͞BMP complex regulated hepcidin expr...

The antimicrobial peptide hepcidin appears to play a central role in the regulation of iron homeostasis. In intact animals, iron overload or the injection of lipopolysaccharide (LPS) stimulates transcription of HAMP, the gene that encodes hepcidin. In isolated hepatocytes, IL-6, an inflammatory cytokine the production of which is stimulated by LPS, up-regulates transcription of hepcidin. In contrast, iron has no stimulatory effect on hepcidin expression in isolated hepatocytes. There is apparently a signaling pathway, activated by iron, that is present in the intact animal but not in isolated hepatocytes. Studies in humans and mice have shown that this iron-dependent pathway requires the presence of Hfe, hemojuvelin, and probably transferrin receptor 2 (tfr-2). To determine whether activation of hepcidin transcription by IL-6 also requires Hfe and tfr-2, we have studied mice homozygous for targeted disruption of HFE, ␤2-microglobulin, and for a truncating mutation of TFR-2. We show that these mutant mice react normally to injection of endotoxin and that their isolated hepatocytes react normally to IL-6. This indicates that the signaling pathway activated by IL-6 does not require either Hfe or tfr-2. Mice with disruption of the gene encoding IL-6 seem to have a blunted response to LPS, but the statistical significance of the small response documented is borderline. It is therefore not clear whether LPS stimulates secretion of cytokines other than IL-6 that may stimulate hepcidin transcription.

Summary Hepcidin, the master regulator of enteric iron absorption, is controlled by the opposing effects of pathways activated in response to iron excess or iron attenuation. Iron excess is regulated through a pathway involving the cell surface receptor hemojuvelin (HFE2) that stimulates expression of the hepcidin encoding gene (HAMP). Iron attenuation is countered through a pathway involving the hepatocyte‐specific plasma membrane protease matriptase‐2 encoded by TMPRSS6, leading to suppression of HAMP expression. The non‐redundant function of hemojuvelin and matriptase‐2 has been deduced from the phenotype imparted by mutations of HFE2 and TMPRSS6, which cause iron excess and iron deficiency respectively. Hemojuvelin is positioned to be the ideal substrate for matriptase‐2. To examine the relationship between hemojuvelin and matriptase‐2 in vivo, we crossed mice lacking the protease domain of matriptase‐2 with mice lacking hemojuvelin. Mice lacking functional matriptase‐2 and hemojuvelin exhibited low Hamp (Hamp1) expression, high serum and liver iron, and high transferrin saturation. Surprisingly, the double mutant mice also exhibited lower levels of iron in the heart compared to hemojuvelin‐deficient mice, demonstrating a possible cardioprotective effect resulting from the loss of matriptase‐2. This phenotype is consistent with hemojuvelin being a major substrate for matriptase‐2/TMPRSS6 protease activity.

Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque rupture process and identify a proximal biomarker for AMI. Thus, we aimed to identify a transcriptomic signature of AMI present in whole blood, but derived from CECs. Candidate genes indicative of AMI were nominated from microarray of enriched CEC samples, and then verified for detectability and predictive potential via qPCR in whole blood. This signature was validated in an independent cohort. Our findings suggest that a whole blood CEC-derived molecular signature identifies patients with AMI and sets the framework to potentially identify the earlier stages of an impending cardiac event when used in concert with clinical history and other diagnostics where conventional biomarkers indicative of myonecrosis remain undetected.