Bone morphogenetic protein (BMP) signaling induces hepatic expression of the peptide hormone hepcidin. Hepcidin reduces serum iron levels by promoting degradation of the iron exporter ferroportin. A relative deficiency of hepcidin underlies the pathophysiology of many of the genetically distinct iron overload disorders, collectively termed hereditary hemochromatosis. Conversely, chronic inflammatory conditions and neoplastic diseases can induce high hepcidin levels, leading to impaired mobilization of iron stores and the anemia of chronic disease. Two BMP type I receptors, Alk2 (Acvr1) and Alk3 (Bmpr1a), are expressed in murine hepatocytes. We report that liver-specific deletion of either Alk2 or Alk3 causes iron overload in mice. The iron overload phenotype was more marked in Alk3-than in Alk2-deficient mice, and Alk3 deficiency was associated with a nearly complete ablation of basal BMP signaling and hepci- IntroductionThe hepatic hormone hepcidin regulates serum iron levels in mice and humans by inducing degradation of the iron exporter, ferroportin. 1,2 Low ferroportin levels reduce intestinal iron absorption and the release of iron from macrophage stores. Human hereditary hemochromatosis is characterized by low hepcidin levels, leading to iron accumulation in liver, heart, and endocrine organs. 1,3 Similarly, hepcidin deficiency causes hepatic iron overload in mice. 2,4 In contrast, high hepcidin levels contribute to the anemia of chronic disease (ACD) by reducing iron bioavailability for erythropoiesis. 5,6 Recent studies have demonstrated a critical role for bone morphogenetic protein (BMP) signaling in the regulation of hepcidin expression by iron. [7][8][9][10][11] Binding of BMP ligands to type I and type II BMP receptors induces the type II receptor to phosphorylate and activate the type I receptor. The activated type I receptor, in turn, phosphorylates intracellular signaling molecules, including SMADs 1, 5, and 8. Phosphorylated SMADs 1, 5, and 8 bind SMAD4 and together translocate to the nucleus, where they activate the expression of genes, including hepcidin and the Id family of transcription factors. 12 Deficiency of Smad4, 10 the BMP coreceptor hemojuvelin, 13,14 or BMP6 15,16 in hepatocytes reduces expression of hepcidin 17,18 and induces iron overload. In addition, BMP signaling appears to have an important role in the induction of hepcidin expression by inflammatory mediators that are involved in ACD. 11,19,20 There are 4 type I BMP receptors: Alk1, Alk2, Alk3, and Alk6. The identity of the type I BMP receptor(s) responsible for iron-dependent signaling and the regulation of hepcidin expression in hepatocytes are unknown. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver, 21 and global Alk6 deficiency does not induce iron overload in mice (D. R. Campagna, P. J. Schmidt, and M.D.F., unpublished observations, January 2011). In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. 21 To identify the type I BMP receptor required for th...
SummaryRNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli 's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of N gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.
Key Points Presence of the BMP type I receptor Alk3 is required for interleukin-6 to induce hepatic hepcidin gene expression. Alk3 contributes to the induction of hypoferremia by interleukin-6.
The bone morphogenetic protein (BMP) type II receptor (BMPR2) has a long cytoplasmic tail domain whose function is incompletely elucidated. Mutations in the tail domain of BMPR2 are found in familial cases of pulmonary arterial hypertension. To investigate the role of the tail domain of BMPR2 in BMP signaling, we generated a mouse carrying a Bmpr2 allele encoding a non-sense mediated decay-resistant mutant receptor lacking the tail domain of Bmpr2. We found that homozygous mutant mice died during gastrulation, whereas heterozygous mice grew normally without developing pulmonary arterial hypertension. Using pulmonary artery smooth muscle cells (PaSMC) from heterozygous mice, we determined that the mutant receptor was expressed and retained its ability to transduce BMP signaling. Heterozygous PaSMCs exhibited a BMP7‑specific gain of function, which was transduced via the mutant receptor. Using siRNA knockdown and cells from conditional knockout mice to selectively deplete BMP receptors, we observed that the tail domain of Bmpr2 inhibits Alk2‑mediated BMP7 signaling. These findings suggest that the tail domain of Bmpr2 is essential for normal embryogenesis and inhibits Alk2‑mediated BMP7 signaling in PaSMCs.
686 Introduction: Anemia of chronic disease (ACD), the second most prevalent form of anemia, is commonly associated with chronic inflammatory, infectious, or neoplastic conditions. ACD is characterized by high hepcidin levels that decrease serum iron levels by inducing degradation of the iron exporter ferroportin. In contrast, a relative deficiency of hepcidin leads to ferroportin overexpression and iron overload. Hepcidin is transcriptionally regulated by interleukin-6 (IL-6) and bone morphogenic protein (BMP) signaling. Binding of BMP ligands to type II and type I BMP receptors induces the type II receptor to phosphorylate and activate one of four type I receptors. We sought to identify the type I BMP receptor that participates in the ability of IL-6 to induce hepatic hepcidin gene expression. Methods: The four type I BMP receptors are Alk1, Alk2, Alk3, and Alk6. Alk1 is predominantly expressed in the endothelium. Alk6 is expressed at low levels in murine liver. In contrast, Alk2 and Alk3 are abundantly expressed in hepatocytes. Global deficiency of Alk2 or Alk3 is embryonic lethal. To selectively delete Alk2 or Alk3 in hepatocytes, we studied mice homozygous for Alk2 or Alk3 sequences flanked by loxP sites (Alk2fl/fl and Alk3fl/fl, respectively) that also carried a transgene specifying Cre recombinase under the control of the albumin gene promoter (Alb-Cre). Eight- to 12-week-old male mice (Alk2fl/fl, Alk2fl/fl; Alb-Cre, Alk3fl/fl, Alk3fl/fl; Alb-Cre) on a standard, iron-replete diet were injected via the tail vein with an adenovirus specifying IL-6 (Ad.IL-6) or an adenovirus specifying green fluorescent protein (GFP; Ad.GFP), as a control (1010 particles per ml for both). Seventy-two hours later, mice were euthanized, and blood was obtained for measurement of serum iron levels and transferrin saturations. Livers were harvested, and RNA was extracted. Hepatic levels of mRNAs encoding Alk2, Alk3, hepcidin, heme oxygenase-1 (HO-1, a transcriptional target of IL-6), and Id-1 (a BMP gene target) were measured by qRT-PCR. Hepatic STAT3 phosphorylation (a marker of IL-6 receptor activation) was measured using immunoblot techniques. Results: Liver-specific deletion of Alk2 or Alk3 caused mild and severe iron overload, respectively. Injection of Ad.IL-6, but not Ad.GFP, decreased serum iron levels and transferrin saturations in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. In contrast, infection of Alk3fl/fl; Alb-Cre mice with Ad.IL-6 did not alter serum iron levels and only modestly reduced transferrin saturations. Infection with Ad.IL-6 induced of hepatic hepcidin gene expression in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice. Hepatic hepcidin mRNA levels were markedly reduced in Ad.GFP-infected Alk3fl/fl; Alb-Cre mice, and infection with Ad.IL-6 failed to increase hepcidin mRNA levels in this genotype. Ad.IL-6 infection induced hepatic Id-1 mRNA levels in Alk2fl/fl, Alk2fl/fl; Alb-Cre, and Alk3fl/fl mice, but not in Alk3fl/fl; Alb-Cre mice. Infection with Ad.IL-6 induced hepatic STAT-3 phosphorylation and HO-1 gene expression in all 4 genotypes of mice. Conclusions: Taken together, these results demonstrate that the response to IL-6, as reflected by STAT-3 phosphorylation and induction of HO-1 gene expression, does not require Alk2 or Alk3. In contrast, BMP signaling, predominantly via Alk3, is essential for the induction of hepcidin gene expression by IL-6. These results suggest that selective inhibition of Alk3 may represent a novel therapeutic approach to the treatment of ACD. Disclosures: Peterson: Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Yu:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties. Bloch:Massachusetts General Hospital: Patents & Royalties, The Massachusetts General Hospital has filed patents related to the use of small molecule inhibitors of BMP signaling to modulate iron metabolism, and PBY, RTP and KDB may be eligible to receive royalties.
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