Iron is essential for many cellular functions; consequently, disturbances of iron homeostasis, leading to either iron deficiency or iron overload, can have significant clinical consequences. Despite the clinical prevalence of these disorders, the mechanism by which dietary iron is absorbed into the body is poorly understood. We have identified a key component in intestinal iron transport by study of the sex-linked anaemia (sla) mouse, which has a block in intestinal iron transport. Mice carrying the sla mutation develop moderate to severe microcytic hypochromic anaemia. Although these mice take up iron from the intestinal lumen into mature epithelial cells normally, the subsequent exit of iron into the circulation is diminished. As a result, iron accumulates in enterocytes and is lost during turnover of the intestinal epithelium. Biochemical studies have failed to identify the underlying difference between sla and normal mice, therefore, we used a genetic approach to identify the gene mutant in sla mice. We describe here a novel gene, Heph, encoding a transmembrane-bound ceruloplasmin homologue that is mutant in the sla mouse and highly expressed in intestine. We suggest that the hephaestin protein is a multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation and that it is an important link between copper and iron metabolism in mammals.

Angiotensin II is an important effector molecule controlling blood pressure and volume in the cardiovascular system. Its importance is manifested by the efficacy of angiotensin-converting enzyme inhibitors in the treatment of hypertension and congestive heart failure. Angiotensin II interacts with two pharmacologically distinct subtypes of cell-surface receptors, AT1 and AT2. AT1 receptors seem to mediate the major cardiovascular effects of angiotensin II. Here we report the isolation by expression cloning of a complementary DNA encoding a unique protein with the pharmacological specificity of a vascular AT1 receptor. Hydropathic modelling of the deduced protein suggests that it shares the seven-transmembrane-region motif with the G protein-coupled receptor superfamily. Knowledge of the AT1 receptor primary sequence should now permit structural analysis, definition of the angiotensin II receptor gene family and delineation of the contribution of AT receptors to the genetic component of hypertension.

Lu X, Murphy TC, Nanes MS, Hart CM. PPAR␥ regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-B. Am J Physiol Lung Cell Mol Physiol 299: L559 -L566, 2010. First published July 9, 2010; doi:10.1152/ajplung.00090.2010.-NADPH oxidases are a major source of superoxide production in the vasculature. The constitutively active Nox4 subunit, which is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension, is highly expressed in vascular wall cells. We demonstrated that rosiglitazone, a synthetic agonist of the peroxisome proliferatoractivated receptor-␥ (PPAR␥), attenuated hypoxia-induced pulmonary hypertension, vascular remodeling, Nox4 induction, and reactive oxygen species generation in the mouse lung. The current study examined the molecular mechanisms involved in PPAR␥-regulated, hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposing HPASMC to 1% oxygen for 72 h increased Nox4 gene expression and H 2O2 production, both of which were reduced by treatment with rosiglitazone during the last 24 h of hypoxia exposure or by treatment with small interfering RNA (siRNA) to Nox4. Hypoxia also increased HPASMC proliferation as well as the activity of a Nox4 promoter luciferase reporter, and these increases were attenuated by rosiglitazone. Chromatin immunoprecipitation assays demonstrated that hypoxia increased binding of the NF-B subunit, p65, to the Nox4 promoter and that binding was attenuated by rosiglitazone treatment. The role of NF-B in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity, whereas siRNA to p50 or p65 attenuated hypoxic stimulation of Nox4 promoter activity. These results provide novel evidence for NF-B-mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPAR␥. These data provide new insights into potential mechanisms by which PPAR␥ activation inhibits Nox4 upregulation and the proliferation of cells in the pulmonary vascular wall to ameliorate pulmonary hypertension and vascular remodeling in response to hypoxia. nuclear factor-B; peroxisome proliferator-activated receptor-␥ NADPH OXIDASES ARE A MAJOR source of superoxide production in the vasculature that contributes to endothelial dysfunction and vascular cell proliferation (4,19). In nonphagocytic cells, the catalytic moiety of NADPH oxidases is composed of one or more gp91 phox (Nox2) homologs, Nox1, -3, -4, or -5, Duox1, or Duox2 (27). These Nox homologs associate with the membrane-bound p22 phox subunit to generate reactive oxygen species (ROS). Nox4 is highly expressed in vascular wall cells including smooth muscle and endothelial cells (47). In contrast to the other Nox homologs, current evidence indicates that Nox4 is constitutively active (1), and increases in Nox4 mRNA levels increase Nox4 activity (45). Nox4 expression is increased by diverse stimuli (4) including E2F transcription factors, serum starvati...

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor g (PPARg) contribute to hypoxiainduced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxiaexposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O 2 ) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARg, and transforming growth factor (TGF)b1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O 2 ) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARg and TGF-b1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H 2 O 2 release, proliferation, and TGF-b1 expression and blunted reductions in PPARg in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-b1 and reductions in PPARg expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxiainduced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.Keywords: rosiglitazone; PPARg; TGF-b; pulmonary hypertension Pulmonary hypertension (PH) is a progressive disorder associated with significant morbidity and mortality. Although recent therapeutic advances have improved survival for patients with PH, the prognosis remains poor (1). The pathobiology of PH is complex, and factors that contribute to endothelial dysfunction have been implicated in pathogenesis (2, 3). Among these factors, NADP reduced (NADPH) oxidase enzymes that produce reactive oxygen species (ROS) contribute to the development of a variety of vascular diseases, such as atherosclerosis (4) and systemic (5) and pulmonary hypertension (6). NADPH oxidases catalyze the reduction of molecular oxygen to generate superoxide (O 2 .2 ), hydrogen peroxide (H 2 O 2 ), or secondary oxidants (7). Seven isoforms of the catalytic moiety of the nonphagocytic NADPH oxidase enzyme have been described (Nox1-5, Duox1-2). These subunits are homologous to the catalytic moiety of the prototype phagocytic NADPH oxidase Nox2 (or gp91 phox ) but differ from each other regarding cellular localization, tissue distribution, regulation, activation, and expression (7,8). For example, although both Nox1 and Nox4 are expressed in vascular smooth muscle cells (VSMCs), they are targeted to discreet intracellular locations, are differe...

Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules such as p53, cyclins, myc, BRCA1, HIF-1a, etc. The Kruppel-like factor 5 (KLF5) transcription factor regulates biological processes involved in carcinogenesis, angiogenesis, and smooth muscle cell differentiation. In carcinogenesis, KLF5's role has been indicated by frequent genetic deletion as well as functional studies. Here we show that KLF5 is an unstable protein with a short half-life. Destruction of KLF5 was prevented by each of the proteasome-specific inhibitors tested but not by an inhibitor for trypsin-like proteases and cysteine proteases or by a lysosome inhibitor in epithelial cells. Furthermore, KLF5 underwent ubiquitination, and deletion of a 56-amino-acid sequence adjacent to a known transactivation domain of KLF5 significantly reduced its ubiquitination and degradation. Interestingly, cancer cells appeared to be more active in KLF5 degradation than untransformed epithelial cells, yet their proteasome activity was not higher. These results suggest that KLF5 protein is degraded at least in part through ubiquitinationproteasome pathway, which may have become hyperactive for KLF5 in cancer cells.

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.CCCH zinc finger ͉ poly(A) binding protein ͉ RNA binding

Background: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. Aims: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. Methods: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive 59 Fe. Results: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. Conclusions: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

The promoter region of the endothelial cell nitric oxide synthase (ecNOS) gene contains potential response elements for transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 plays an important role in the pathogenesis of atherosclerosis, vascular hypertrophy, and angiogenesis. We therefore sought to determine whether TGF beta 1 might modulate ecNOS expression in bovine aortic endothelial cells (BAEC). TGF beta 1 increased ecNOS mRNA in a dose-dependent manner. TGF beta 1 also increased ecNOS protein content. The production of nitrogen oxides (NOx), assessed by chemiluminescence, and nitric oxide synthase activity, assessed by arginine/citrulline conversion were increased in TGF beta 1-treated cells. Transcriptional activity of the 5'-flanking promoter region of the ecNOS gene was increased by TGF beta 1, as assessed by transfection with promoter/luciferase constructs. Deletion analysis suggested that the TGF beta 1-response element was present between nucleotides -1269 and -935 from the first transcription start site, in which a putative nuclear factor-1 (NF-1) binding site existed. Gel shift assays showed that nuclear protein(s), immunologically similar to CCAAT transcription factor/NF-1, bound to the putative NF-1 binding site in a sequence-specific manner. Mutation of the putative NF-1 binding site in the promoter/luciferase construct significantly decreased the responsiveness to TGF beta 1. In conclusion, TGF beta 1 increases ecNOS expression associated with an increase in production of NO in BAEC. This response is probably mediated by transcriptional activation of the ecNOS gene promoter.