Smad7 deficiency decreases iron and haemoglobin through hepcidin up‐regulation by multilayer compensatory mechanisms

To maintain iron homoeostasis, the iron regulatory hormone hepcidin is tightly controlled by BMP‐Smad signalling pathway, but the physiological role of Smad7 in hepcidin regulation remains elusive. We generated and characterized hepatocyte‐specific Smad7 knockout mice (Smad7^Alb/Alb), which showed decreased serum iron, tissue iron, haemoglobin concentration, up‐regulated hepcidin and increased phosphor‐Smad1/5/8 levels in both isolated primary hepatocytes and liver tissues. Increased levels of hepcidin lead to reduced expression of intestinal ferroportin and mild iron deficiency anaemia. Interestingly, we found no difference in hepcidin expression or phosphor‐Smad1/5/8 levels between iron‐challenged Smad7^Alb/Alb and Smad7^flox/flox, suggesting other factors assume the role of iron‐induced hepcidin regulation in Smad7 deletion. We performed RNA‐seq to identify differentially expressed genes in the liver. Significantly up‐regulated genes were then mapped to pathways, revealing TGF‐β signalling as one of the most relevant pathways, including the up‐regulated genes Smad6, Bambi and Fst (Follistatin). We found that Smad6 and Bambi—but not Follistatin—are controlled by the iron‐BMP–Smad pathway. Overexpressing Smad6, Bambi or Follistatin in cells significantly reduced hepcidin expression. Smad7 functions as a key regulator of iron homoeostasis by negatively controlling hepcidin expression, and Smad6 and Smad7 have non‐redundant roles. Smad6, Bambi and Follistatin serve as additional inhibitors of hepcidin in the liver.     

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.     

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.     

The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin

The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation, and is downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2(MASK)), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2(MASK) shows no cleavage activity and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2(MASK). The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.     

Matriptase-2- and proprotein convertase-cleaved forms of hemojuvelin have different roles in the down-regulation of hepcidin expression

Hemojuvelin (HJV) is an important regulator of iron metabolism. Membrane-anchored HJV up-regulates expression of the iron regulatory hormone, hepcidin, through the bone morphogenic protein (BMP) signaling pathway by acting as a BMP co-receptor. HJV can be cleaved by the furin family of proprotein convertases, which releases a soluble form of HJV that suppresses BMP signaling and hepcidin expression by acting as a decoy that competes with membrane HJV for BMP ligands. Recent studies indicate that matriptase-2 binds and degrades HJV, leading to a decrease in cell surface HJV. In the present work, we show that matriptase-2 cleaves HJV at Arg(288), which produces one major soluble form of HJV. This shed form of HJV has decreased ability to bind BMP6 and does not suppress BMP6-induced hepcidin expression. These results suggest that the matriptase-2 and proprotein convertase-cleavage products have different roles in the regulation of hepcidin expression.     

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv^−/−) mice that have severe iron overload and marked suppression of hepcidin expression, we detected 4-fold higher hepatic BMP6 mRNA than in wild-type counterparts. These results indicate that Hjv^−/− mice do not lack BMP6. Furthermore, iron depletion in Hjv^−/− mice decreased hepatic BMP6 mRNA. Expression of HJV in hepatocytes of Hjv^−/− mice using an AAV2/8 vector, increased hepatic hepcidin mRNA by 65-fold and phosphorylated Smad1/5/8 in the liver by about 2.5-fold. However, no significant change in BMP6 mRNA was detected in either the liver or the small intestine of these animals. Our results revealed a close correlation of hepatic BMP6 mRNA expression with hepatic iron-loading. Together, our data indicate that the regulation of hepatic BMP6 expression by iron is independent of HJV, and that expression of HJV in hepatocytes plays an essential role in hepcidin expression by potentiating the BMP6-mediated signaling.     

The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+‐dependent succinic semialdehyde dehydrogenase

Lower plant species including some green algae, non‐vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP⁺‐dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ‐aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD⁺ is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP⁺ binding induces a conformational change of the loop carrying Arg‐228, which seals the NADP⁺ in the coenzyme cavity via its 2′‐phosphate and α‐phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg‐121 and Arg‐457, and a hydrogen bond with Tyr‐296. While both arginine residues are pre‐formed for substrate/product binding, Tyr‐296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5.     

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down‐regulated by alcohol in cell lines and animal models. This down‐regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real‐time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down‐regulation of hepcidin expression leading to up‐regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.     

In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7A^rec) was compared with the native fungal enzyme (TrCel7A^nat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7A^rec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7A^rec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7A^rec had 25% lower catalytic activity than TrCel7A^nat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7A^rec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.     

Loss of GluN2A‐containing NMDA receptors impairs extra‐dimensional set‐shifting

Glutamate neurotransmission via the N‐methyl‐d ‐aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re‐learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non‐discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2A^KO), heterozygous (GluN2A^HET) and wild‐type (WT) littermates on an attentional set‐shifting task using species‐specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2A^KO were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2A^KO showed a specific and significant impairment in ventromedial prefrontal cortex‐mediated set‐shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands.     

Molecular diversity and landscape genomics of the crop wild relative Triticum urartu across the Fertile Crescent

Modern plant breeding can benefit from the allelic variation that exists in natural populations of crop wild relatives that evolved under natural selection in varying pedoclimatic conditions. In this study, next‐generation sequencing was used to generate 1.3 million genome‐wide single nucleotide polymorphisms (SNPs) on ex situ collections of Triticum urartu L., the wild donor of the A^u subgenome of modern wheat. A set of 75 511 high‐quality SNPs were retained to describe 298 T. urartu accessions collected throughout the Fertile Crescent. Triticum urartu showed a complex pattern of genetic diversity, with two main genetic groups distributed sequentially from west to east. The incorporation of geographical information on sampling points showed that genetic diversity was correlated to the geographical distance (R² = 0.19) separating samples from Jordan and Lebanon, from Syria and southern Turkey, and from eastern Turkey, Iran and Iraq. The wild emmer genome was used to derive the physical positions of SNPs on the seven chromosomes of the A^u subgenome, allowing us to describe a relatively slow decay of linkage disequilibrium in the collection. Outlier loci were described on the basis of the geographic distribution of the T. urartu accessions, identifying a hotspot of directional selection on chromosome 4A. Bioclimatic variation was derived from grid data and related to allelic variation using a genome‐wide association approach, identifying several marker–environment associations (MEAs). Fifty‐seven MEAs were associated with altitude and temperature measures while 358 were associated with rainfall measures. The most significant MEAs and outlier loci were used to identify genomic loci with adaptive potential (some already reported in wheat), including dormancy and frost resistance loci. We advocate the application of genomics and landscape genomics on ex situ collections of crop wild relatives to efficiently identify promising alleles and genetic materials for incorporation into modern crop breeding.     

Iron-mediated retinal degeneration in haemojuvelin-knockout mice

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE (human leucocyte antigen-like protein involved in iron homoeostasis), transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Recent studies have established the expression of all of the five genes in the retina, indicating their importance in retinal iron homoeostasis. Previously, we demonstrated that HJV is expressed in RPE (retinal pigment epithelium), the outer and inner nuclear layers and the ganglion cell layer. In the present paper, we report on the consequences of Hjv deletion on the retina in mice. Hjv-/- mice at ≥18 months of age had increased iron accumulation in the retina with marked morphological damage compared with age-matched controls; these changes were not found in younger mice. The retinal phenotype in Hjv-/- mice included hyperplasia of RPE. We isolated RPE cells from wild-type and Hjv-/- mice and examined their growth patterns. Hjv-/- RPE cells were less senescent and exhibited a hyperproliferative phenotype. Hjv-/- RPE cells also showed up-regulation of Slc7a11 (solute carrier family 7 member 11 gene), which encodes the 'transporter proper' subunit xCT in the heterodimeric amino acid transporter xCT/4F2hc (cystine/glutamate exchanger). BMP6 (bone morphogenetic protein 6) could not induce hepcidin expression in Hjv-/- RPE cells, confirming that retinal cells require HJV for induction of hepcidin via BMP6 signalling. HJV is a glycosylphosphatidylinositol-anchored protein, and the membrane-associated HJV is necessary for BMP6-mediated activation of hepcidin promoter in RPE cells. Taken together, these results confirm the biological importance of HJV in the regulation of iron homoeostasis in the retina and in RPE.     

Essential Oil Variability in Natural Populations of Artemisia campestris (L.) and Artemisia herba‐alba (Asso) and Incidence on Antiacetylcholinesterase and Antioxidant Activities

The intraspecific variability of Artemisia herba‐alba and A. campestris essential oils and the evaluation of their antioxidant and antiacetylcholinesterase activities were determined. Artemisia herba‐alba essential oil was found rich in camphor (19.61%), α‐thujone (19.40%), β‐thujone (9.44%), chrysanthenone (9.26%), and trans‐sabinyl acetate (8.43%). The major compounds of A. campestris essential oil were germacrene D (16.38%), β‐pinene (16.33%), and limonene (9.17%). Significant variation in the essential oil composition was observed among populations of each species. The divergence between populations was attributed to the variation of some climatic factors such as altitude, annual rainfall, winter cold stress, summer precipitation, summer drought stress, evapotranspiration, and humidity. Artemisia herba‐alba and A. campestris essential oils exhibited promising antioxidant and antiacetylcholinesterase activities. The level of activity varied significantly according to the species and the essential oil. The highest scavenging activity (IC₅₀ = 0.14 mg/ml) and the uppermost capacity to prevent β‐carotene bleaching (IC₅₀ = 0.10 mg/ml) characterized A. campestris from population 6. A. campestris population 3 possessed the uppermost ability to reduce ferric ions (450.7 μmol Fe²⁺/g EO). The population 2 of A. campestris showed the strongest antiacetylcholinesterase activity (IC₅₀ = 0.02 mg/ml). The variation of these activities between the essential oils was explained by their composition differences.     

Down‐regulation of BnDA1, whose gene locus is associated with the seeds weight,improves the seeds weight and organ size in Brassica napus

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down‐regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down‐regulated in B. napus by over expressed of AtDA1^R358K, which is a functional deficiency of DA1 with an arginine‐to‐lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.     

Polymorphism in the neurofibromin gene,Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster

In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS‐specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1‐insertion‐A and Nf1‐deletion‐G. These haplotypes are characterized by a 45‐base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1‐insertion‐A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1‐insertion‐A haplotype is negatively associated with wing size. We found that the Nf1‐insertion‐A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1‐insertion‐A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.     

TaDA1, a conserved negative regulator of kernel size,has an additive effect with TaGW2 in common wheat (Triticum aestivum L.)

Kernel size is an important trait determining cereal yields. In this study, we cloned and characterized TaDA1, a conserved negative regulator of kernel size in wheat (Triticum aestivum). The overexpression of TaDA1 decreased the size and weight of wheat kernels, while its down‐regulation using RNA interference (RNAi) had the opposite effect. Three TaDA1‐A haplotypes were identified in Chinese wheat core collections, and a haplotype association analysis showed that TaDA1‐A‐HapI was significantly correlated with the production of larger kernels and higher kernel weights in modern Chinese cultivars. The haplotype effect resulted from a difference in TaDA1‐A expression levels between genotypes, with TaDA1‐A‐HapI resulting in lower TaDA1‐A expression levels. This favourable haplotype was found having been positively selected during wheat breeding over the last century. Furthermore, we demonstrated that TaDA1‐A physically interacts with TaGW2‐B. The additive effects of TaDA1‐A and TaGW2‐B on kernel weight were confirmed not only by the phenotypic enhancement arising from the simultaneous down‐regulation of TaDA1 and TaGW2 expression, but also by the combinational haplotype effects estimated from multi‐environment field data from 348 wheat cultivars. A comparative proteome analysis of developing transgenic and wild‐type grains indicated that TaDA1 and TaGW2 are involved in partially overlapping but relatively independent protein regulatory networks. Thus, we have identified an important gene controlling kernel size in wheat and determined its interaction with other genes regulating kernel weight, which could have beneficial applications in wheat breeding.