Yisrael Sidis

Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression

Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2−/− hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.

Modulation of bone morphogenetic protein signaling in vivo regulates systemic iron balance

Systemic iron balance is regulated by hepcidin, a peptide hormone secreted by the liver. By decreasing cell surface expression of the iron exporter ferroportin, hepcidin decreases iron absorption from the intestine and iron release from reticuloendothelial stores. Hepcidin excess has been implicated in the pathogenesis of anemia of chronic disease, while hepcidin deficiency has a key role in the pathogenesis of the iron overload disorder hemochromatosis. We have recently shown that hemojuvelin is a coreceptor for bone morphogenetic protein (BMP) signaling and that BMP signaling positively regulates hepcidin expression in liver cells in vitro. Here we show that BMP-2 administration increases hepcidin expression and decreases serum iron levels in vivo. We also show that soluble hemojuvelin (HJV.Fc) selectively inhibits BMP induction of hepcidin expression in vitro and that administration of HJV.Fc decreases hepcidin expression, increases ferroportin expression, mobilizes splenic iron stores, and increases serum iron levels in vivo. These data support a role for modulators of the BMP signaling pathway in treating diseases of iron overload and anemia of chronic disease.

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling.

Activins, inhibins, and follistatins: from endocrinology to signaling. A paradigm for the new millennium.

It has been 70 years since the name inhibin was used to describe a gonadal factor that negatively regulated pituitary hormone secretion. The majority of this period was required to achieve purification and definitive characterization of inhibin, an event closely followed by identification and characterization of activin and follistatin (FS). In contrast, the last 15–20 years saw a virtual explosion of information regarding the biochemistry, physiology, and biosynthesis of these proteins, as well as identification of activin receptors, and a unique mechanism for FS action—the nearly irreversible binding and neutralization of activin. Many of these discoveries have been previously summarized; therefore, this review will cover the period from the mid 1990s to present, with particular emphasis on emerging themes and recent advances. As the field has matured, recent efforts have focused more on human studies, so the endocrinology of inhibin, activin, and FS in the human is summarized first. Another area receiving significant recent attention is local actions of activin and its regulation by both FS and inhibin. Because activin and FS are produced in many tissues, we chose to focus on a few particular examples with the most extensive experimental support, the pituitary and the developing follicle, although nonreproductive actions of activin and FS are also discussed. At the cellular level, it now seems that activin acts largely as an autocrine and/or paracrine growth factor, similar to other members of the transforming growh factor β superfamily. As we discuss in the next section, its actions are regulated extracellularly by both inhibin and FS. In the final section, intracellular mediators and modulators of activin signaling are reviewed in detail. Many of these are shared with other transforming growh factor β superfamily members as well as unrelated molecules, and in a number of cases, their physiological relevance to activin signal propagation remains to be elucidated. Nevertheless, taken together, recent findings suggest that it may be more appropriate to consider a new paradigm for inhibin, activin, and FS in which activin signaling is regulated extracellularly by both inhibin and FS whereas a number of intracellular proteins act to modulate cellular responses to these activin signals. It is therefore the balance between activin and all of its modulators, rather than the actions of any one component, that determines the final biological outcome. As technology and model systems become more sophisticated in the next few years, it should become possible to test this concept directly to more clearly define the role of activin, inhibin, and FS in reproductive physiology.

DRAGON: a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)β superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGFβ ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGFβ signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGFβ ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling.

Hemojuvelin regulates hepcidin expression via a selective subset of BMP ligands and receptors independently of neogenin

Hemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.

Mutations in Prokineticin 2 and Prokineticin receptor 2genes in Human Gonadotrophin-Releasing Hormone Deficiency: Molecular Genetics and Clinical Spectrum

CONTEXT Mice deficient in prokineticin 2(PROK2) and prokineticin receptor2 (PROKR2) exhibit variable olfactory bulb dysgenesis and GnRH neuronal migration defects reminiscent of human GnRH deficiency. OBJECTIVES We aimed to screen a large cohort of patients with Kallmann syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (IHH) for mutations in PROK2/PROKR2, evaluate their prevalence, define the genotype/phenotype relationship, and assess the functionality of these mutant alleles in vitro. DESIGN Sequencing of the PROK2 and PROKR2 genes was performed in 170 KS patients and 154 nIHH. Mutations were examined using early growth response 1-luciferase assays in HEK 293 cells and aequorin assays in Chinese hamster ovary cells. RESULTS Four heterozygous and one homozygous PROK2 mutation (p.A24P, p.C34Y, p.I50M, p.R73C, and p.I55fsX1) were identified in five probands. Four probands had KS and one nIHH, and all had absent puberty. Each mutant peptide impaired receptor signaling in vitro except the I50M. There were 11 patients who carried a heterozygous PROKR2 mutation (p.R85C, p.Y113H, p.V115M, p.R164Q, p.L173R, p.W178S, p.S188L, p.R248Q, p.V331M, and p.R357W). Among them, six had KS, four nIHH, and one KS proband carried both a PROKR2 (p.V115M) and PROK2 (p.A24P) mutation. Reproductive phenotypes ranged from absent to partial puberty to complete reversal of GnRH deficiency after discontinuation of therapy. All mutant alleles appear to decrease intracellular calcium mobilization; seven exhibited decreased MAPK signaling, and six displayed decreased receptor expression. Nonreproductive phenotypes included fibrous dysplasia, sleep disorder, synkinesia, and epilepsy. Finally, considerable variability was evident in family members with the same mutation, including asymptomatic carriers. CONCLUSION Loss-of-function mutations in PROK2 and PROKR2 underlie both KS and nIHH.

A Genetic Basis for Functional Hypothalamic Amenorrhea

BACKGROUND Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. METHODS We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. RESULTS Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been previously shown for PROKR2 L173R and GNRHR R262Q. CONCLUSIONS Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism are found in women with hypothalamic amenorrhea, suggesting that these mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations provide evidence for the role of rare variants in common multifactorial disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00494169.).

FSTL3 deletion reveals roles for TGF-β family ligands in glucose and fat homeostasis in adults

Activin and myostatin are related members of the TGF-β growth factor superfamily. FSTL3 (Follistatin-like 3) is an activin and myostatin antagonist whose physiological role in adults remains to be determined. We found that homozygous FSTL3 knockout adults developed a distinct group of metabolic phenotypes, including increased pancreatic islet number and size, β cell hyperplasia, decreased visceral fat mass, improved glucose tolerance, and enhanced insulin sensitivity, changes that might benefit obese, insulin-resistant patients. The mice also developed hepatic steatosis and mild hypertension but exhibited no alteration of muscle or body weight. This combination of phenotypes appears to arise from increased activin and myostatin bioactivity in specific tissues resulting from the absence of the FSTL3 antagonist. Thus, the enlarged islets and β cell number likely result from increased activin action. Reduced visceral fat is consistent with a role for increased myostatin action in regulating fat deposition, which, in turn, may be partly responsible for the enhanced glucose tolerance and insulin sensitivity. Our results demonstrate that FSTL3 regulation of activin and myostatin is critical for normal adult metabolic homeostasis, suggesting that pharmacological manipulation of FSTL3 activity might simultaneously reduce visceral adiposity, increase β cell mass, and improve insulin sensitivity.

Repulsive guidance molecule (RGMa) alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531–539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820–29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122–14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4 ± 0.2 and 1.4 ± 0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.