Dragon (repulsive guidance molecule b) inhibits IL-6 expression in macrophages.

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling.

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

Patterning molecules; multitasking in the nervous system.

Classical patterning molecules that were previously implicated in controlling cell fate choices in the nervous system have recently been shown to play additional roles in axon guidance and synaptogenesis. Bone morphogenetic proteins (BMPs), Sonic hedgehog (Shh), Wnts, and fibroblast growth factors (FGFs) all participate in multiple acts of controlling neuronal circuit assembly. Depending on the cellular context, they can provide instructive signals at the growth cone or synapse or alternatively can elicit responses in the nucleus initiating transcriptional changes. Differences in signal transduction pathways may contribute to the diversity of the functional repertoire of these versatile molecules.

Deferasirox reduces iron overload in a murine model of juvenile hemochromatosis.

Mutations in hemojuvelin (HJV) cause severe juvenile hemochromatosis, characterized by iron loading of the heart, liver, and pancreas. Knockout (KO) mice lacking HJV (Hjv-/-) spontaneously load with dietary iron and, therefore, present a model for hereditary hemochromatosis (HH). In HH, iron chelation may be considered in noncandidates for phlebotomy. We examined the effects of deferasirox, an oral chelator, in Hjv-/- mice. Hepatic, cardiac, splenic, and pancreatic iron were determined by measuring elemental iron and scoring histological sections. Heart and liver iron levels were also determined repeatedly by quantitative R2* magnetic resonance imaging (MRI). The time course of iron loading without intervention was followed from Week 8 of age (study start) to Week 20, when once-daily (5x/week) deferasirox was administered, to Week 28. At 8 weeks, liver iron of KO mice was already markedly elevated versus wild-type mice (P<0.001) and reached a plateau around Week 14. In contrast, Week 8 cardiac and pancreatic iron levels were similar in both KO and wild-type mice and, compared with the liver, showed a delayed but massive iron loading up to Week 20. Contrary to the liver, heart, and pancreas, the KO mice spleen had lower iron content versus wild-type mice. In Hjv-/- mice, liver and heart iron burden was effectively reduced with deferasirox 100 mg/kg (P<0.05). Although deferasirox was less efficacious at this dose in the pancreas, over the observed time period, a clear trend toward reduced organ iron load was noted. There was no noticeable effect of deferasirox upon splenic iron in Hjv-/- mice. Quantitative R2* MRI demonstrated the ability to assess iron concentrations in the liver and myocardial muscle accurately and repetitively. Hepatic (R=0.86; P=3.2*10(-12)) and delayed myocardial (R=0.81; P=2.9*10(-10)) iron accumulation could be followed noninvasively with high agreement to invasive methods.

Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload.

Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.

Repulsive guidance molecule (RGM) gene function is required for neural tube closure but not retinal topography in the mouse visual system.

The establishment of topographic projections in the developing visual system depends on the spatially and temporally controlled expression of axon guidance molecules. In the developing chick tectum, the graded expression of the repulsive guidance molecule (RGM) has been proposed to be involved in controlling the topography of the retinal ganglion cell (RGC) axon termination zones along the anteroposterior axis of the tectum. We now show that there are three mouse proteins homologous to chick RGM displaying similar proteolytic processing but exhibiting differential cell-surface targeting by glycosyl phosphatidylinositol anchor addition. Two members of this gene family (mRGMa and mRGMb) are expressed in complementary patterns in the nervous system, and mRGMa is expressed prominently in the superior colliculus at the time of anteroposterior targeting of RGC axons. The third member of the family (mRGMc) is expressed almost exclusively in skeletal muscles. Functional studies in the mouse reveal a role for mRGMa in controlling cephalic neural tube closure, thus defining an unexpected role for mRGMa in early embryonic development. In contrast, mRGMa mutant mice did not exhibit defects in anteroposterior targeting of RGC axons to their stereotypic termination zones in the superior colliculus.

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro.

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.

Studying gene expression in bone by in situ hybridization.

Here, we described a method for carrying out nonradioactive in situ hybridization to detect mRNA transcripts in cryostat sections of mouse bone using the CryoJane(®) Tape-Transfer System and digoxigenin (DIG)-labeled riboprobes.

Uncommon or delayed adverse events associated with imatinib treatment for chronic myeloid leukemia.

Imatinib, a tyrosine kinase inhibitor, is the first-line therapy for chronic myeloid leukemia (CML). The majority of patients continue treatment for their lifespan because discontinuation generally results in relapse. Many patients treated with imatinib experience adverse events (AEs) at some time during their treatment. Commonly encountered AEs and their management are well known. However, in addition to the common AEs with imatinib, there is a significant number of patients who display either uncommon or delayed AEs. These events can involve cardiac, renal, or dermatologic problems, and fluid retention. Herein, we review these less-than-common side effects and the hazard of administering imatinib during pregnancy. While chronic treatment with imatinib has revolutionized CML prognosis, physicians should be aware of both the common and uncommon adverse reactions.

Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone.

The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.

A rapid, nonradioactive in situ hybridization technique for use on cryosectioned adult mouse bone.

In situ hybridization (ISH) of adult bone is a difficult task that requires at least 3-5 weeks for decalcification, paraffin embedding, and sectioning. For that reason, bone ISH is often done only on embryonic or newborn animal tissue, leaving unanswered the question of gene expression in adults. Here, we report the development of an ISH system that requires only 7 days for acid-free decalcification, embedding, and sectioning, conditions that are conducive to preservation of tissue mRNA. The tissue cryosections, derived from adult mice 3-12 weeks old, were cut using the CryoJane Tape Transfer system. Paraffin-sectioned and cryosectioned tissue have comparable morphology. Examples are given of cryosections that were hybridized and stained enzymatically with digoxigenin-labeled riboprobes for mRNA found in either bone-forming osteoblasts (type I collagen, osteocalcin, Runx2) or the hypertrophic or proliferating chondrocytes (type X collagen, Runx2).

Effect of phlebotomy on hepcidin expression in hemojuvelin-mutant mice.

Hemojuvelin (Hjv) is an essential component of the pathway regulating hepcidin (Hamp1) gene expression. Mice with targeted disruption of the Hjv gene (Hjv-/- mice) fail to upregulate hepatic Hamp1 expression following iron overload. The main aim of the study was to determine whether the Hjv protein is also necessary for Hamp1 downregulation. In addition, sex differences in Hamp1 expression in Hjv-/- mice were also examined. Male and female Hjv-/- mice (129SvJ background) were used for the experiments, tissue Hamp1 and Hamp2 mRNA content was determined by real-time PCR. Hepatic Hamp1 mRNA content in male Hjv-/- mice was low (0.6% of Hjv+/+ males), however, female Hjv-/- mice displayed only moderately reduced (to 17%) Hamp1 mRNA levels. Hepatic non-heme iron concentration was similar in Hjv-/- mice of both sexes. Disruption of the Hjv gene did not affect Hamp1 mRNA content in the myocardium or Hamp2 mRNA content in the pancreas. Single phlebotomy resulted in significant reduction of Hamp1 mRNA in both male and female Hjv+/+ mice (to 17% and 27% of controls respectively), measured 20 h after treatment. In Hjv-/- mice, phlebotomy decreased Hamp1 mRNA content to 46% in males and to 11% in females. Bleeding also significantly decreased (to 16%) hepatic Hamp2 mRNA levels in Hjv-/- females. The obtained results indicate that the pathway mediating hepcidin downregulation by phlebotomy does not require functional hemojuvelin protein. In addition, they confirm a significant effect of sex on hepcidin gene expression.