Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function

Alterations in serotonin (5-hydroxytriptamine, 5-HT), norepinephrine, and γ-aminobutyric acid have been linked to the pathophysiology of anxiety and depression, and medications that modulate these neurotransmitters are widely used to treat mood disorders. Recently, the neuropeptide substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), have been proposed as possible targets for new antidepressant and anxiolytic therapies. However, animal and human studies have so far failed to provide a clear consensus on the role of SP in the modulation of emotional states. Here we show that both genetic disruption and acute pharmacological blockade of the NK1R in mice result in a marked reduction of anxiety and stress-related responses. These behavioral changes are paralleled by an increase in the firing rate of 5-HT neurons in the dorsal raphe nucleus, a major source of serotonergic input to the forebrain. NK1R disruption also results in a selective desensitization of 5-HT1A inhibitory autoreceptors, which resembles the effect of sustained antidepressant treatment. Together these results indicate that the SP system powerfully modulates anxiety and suggest that this effect is at least in part mediated by changes in the 5-HT system.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Mechanism of topology simplification by type II DNA topoisomerases

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma

DPC4 is known to mediate signals initiated by type β transforming growth factor (TGFβ) as well as by other TGFβ superfamily ligands such as activin and BMP (bone morphogenic proteins), but mutational surveys of such non-TGFβ receptors have been negative to date. Here we describe the gene structure and novel somatic mutations of the activin type I receptor, ACVR1B, in pancreatic cancer. ACVR1B has not been described previously as a mutated tumor-suppressor gene.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

IL-1-induced NFκB and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK)

Mutant I1A cells, lacking IL-1 receptor-associated kinase (IRAK) mRNA and protein, have been used to study the involvement of IRAK in NFκB and c-Jun N-terminal kinase (JNK) activation. A series of IRAK deletion constructs were expressed in I1A cells, which were then tested for their ability to respond to IL-1. Both the N-terminal death domain and the C-terminal region of IRAK are required for IL-1-induced NFκB and JNK activation, whereas the N-proximal undetermined domain is required for the activation of NFκB but not JNK. The phosphorylation and ubiquitination of IRAK deletion mutants correlate tightly with their ability to activate NFκB in response to IL-1, but IRAK can mediate IL-1-induced JNK activation without being phosphorylated. These studies reveal that the IL-1-induced signaling pathways leading to NFκB and JNK activation diverge either at IRAK or at a point nearer to the receptor.

Involvement of the Ubiquitin/Proteasome System in Sorting of the Interleukin 2 Receptor β Chain to Late Endocytic Compartments

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor β chain (IL2Rβ) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rβ was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rβ, with little effect on its internalization. In addition, we show that a fraction of IL2Rβ can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rβ targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rβ were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rβ toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Characterisation of the gene encoding type II DNA topoisomerase from Leishmania donovani: a key molecular target in antileishmanial therapy

The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5′-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at –639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at ∼132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.

Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue.

The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor alpha on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 1 beta, or tumor necrosis factor alpha augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt-Jakob disease in mice.

The ultrastructural pathology of myelinated axons in mice infected experimentally with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus is characterized by myelin sheath vacuolation that closely resembles that induced in murine spinal cord organotypic cultures by tumor necrosis factor alpha (TNF-alpha), a cytokine produced by astrocytes and macrophages. To clarify the role of TNF-alpha in experimental CJD, we investigated the expression of TNF-alpha in brain tissues from CJD virus-infected mice at weekly intervals after inoculation by reverse transcription-coupled PCR, Northern and Western blot analyses, and immunocytochemical staining. Neuropathological findings by electron microscopy, as well as expression of interleukin 1 alpha and glial fibrillary acidic protein, were concurrently monitored. As determined by reverse transcription-coupled PCR, the expression of TNF-alpha, interleukin 1 alpha, and glial fibrillary acidic protein was increased by approximately 200-fold in the brains of CJD virus-inoculated mice during the course of disease. By contrast, beta-actin expression remained unchanged. Progressively increased expression of TNF-alpha in CJD virus-infected brain tissues was verified by Northern and Western blot analyses, and astrocytes in areas with striking myelin sheath vacuolation were intensely stained with an antibody against murine TNF-alpha. The collective findings of TNF-alpha overexpression during the course of clinical disease suggest that TNF-alpha may mediate the myelin sheath vacuolation observed in experimental CJD.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.