Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation.

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter-driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

Increased salt consumption induces body water conservation and decreases fluid intake.

BACKGROUND: The idea that increasing salt intake increases drinking and urine volume is widely accepted. We tested the hypothesis that an increase in salt intake of 6 g/d would change fluid balance in men living under ultra-long-term controlled conditions. METHODS: Over the course of 2 separate space flight simulation studies of 105 and 205 days' duration, we exposed 10 healthy men to 3 salt intake levels (12, 9, or 6 g/d). All other nutrients were maintained constant. We studied the effect of salt-driven changes in mineralocorticoid and glucocorticoid urinary excretion on day-to-day osmolyte and water balance. RESULTS: A 6-g/d increase in salt intake increased urine osmolyte excretion, but reduced free-water clearance, indicating endogenous free water accrual by urine concentration. The resulting endogenous water surplus reduced fluid intake at the 12-g/d salt intake level. Across all 3 levels of salt intake, half-weekly and weekly rhythmical mineralocorticoid release promoted free water reabsorption via the renal concentration mechanism. Mineralocorticoid-coupled increases in free water reabsorption were counterbalanced by rhythmical glucocorticoid release, with excretion of endogenous osmolyte and water surplus by relative urine dilution. A 6-g/d increase in salt intake decreased the level of rhythmical mineralocorticoid release and elevated rhythmical glucocorticoid release. The projected effect of salt-driven hormone rhythm modulation corresponded well with the measured decrease in water intake and an increase in urine volume with surplus osmolyte excretion. CONCLUSION: Humans regulate osmolyte and water balance by rhythmical mineralocorticoid and glucocorticoid release, endogenous accrual of surplus body water, and precise surplus excretion. FUNDING: Federal Ministry for Economics and Technology/DLR; the Interdisciplinary Centre for Clinical Research; the NIH; the American Heart Association (AHA); the Renal Research Institute; and the TOYOBO Biotechnology Foundation. Food products were donated by APETITO, Coppenrath und Wiese, ENERVIT, HIPP, Katadyn, Kellogg, Molda, and Unilever.

Early detection of NSCLC with scFv selected against IgM autoantibody.

Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88 (95%CI, 0.76-1.0) as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 (95% CI, 0.59-0.85). The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease.

Loss of polymeric immunoglobulin receptor expression is associated with lung tumourigenesis.

Polymeric immunoglobulin receptor (pIgR) expression is downregulated in lung cancer, but its implications in lung tumourigenesis remain unknown. We hypothesised that loss of pIgR expression occurs early, and is associated with cell proliferation and poor prognosis. pIgR expression was evaluated by immunohistochemistry in airways of patients with normal mucosa, pre-invasive lesions and invasive lesions, and correlated with clinical outcomes. 16-HBE and A549 cells stably transfected with pIgR were tested for proliferation, apoptosis and cell cycle progression. Immunostaining was strong in normal epithelium, but severely reduced in pre-invasive lesions and most lung cancers. Persistent expression was associated with younger age and adenocarcinoma subtype but not survival. pIgR overexpression significantly reduced A549 and 16-HBE proliferation. Growth inhibition was not due to cell cycle arrest, increased apoptosis or endoplasmic reticulum stress, but we observed altered expression of genes encoding for membrane proteins, including NOTCH3. Interestingly, NOTCH3 expression was inversely correlated with pIgR expression in cell lines and tissues. pIgR expression was lost in most lung cancers and pre-invasive bronchial lesions, suggesting that pIgR downregulation is an early event in lung tumourigenesis. pIgR overexpression in A549 and 16-HBE cells inhibited proliferation. Future investigations are required to determine the mechanisms by which pIgR contributes to cell proliferation.

Lipopolysaccharide-dependent interaction between PU.1 and c-Jun determines production of lipocalin-type prostaglandin D synthase and prostaglandin D2 in macrophages.

Previously, we reported that expression of lipocalin-prostaglandin D synthase (L-PGDS) is inducible in macrophages and protects from Pseudomonas pneumonia. Here, we investigated the mechanism by which L-PGDS gene expression is induced in macrophages. A promoter analysis of the murine L-PGDS promoter located a binding site of PU.1, a transcription factor essential for macrophage development and inflammatory gene expression. A chromatin immunoprecipitation assay showed that PU.1 bound to the cognate site in the endogenous L-PGDS promoter in response to LPS. Overexpression of PU.1, but not of PU.1(S148A), a mutant inert to casein kinase II (CKII) or NF-kappaB-inducing kinase (NIK), induced L-PGDS in RAW 264.7 cells. Conversely, siRNA silencing of PU.1 expression blunted productions of L-PGDS and prostaglandin D2 (PGD(2)). LPS treatment induced formation of the complex of PU.1 and cJun on the PU.1 site, but inactivation of cJun by treatment with JNK or p38 kinase inhibitor abolished the complex, and suppressed PU.1 transcriptional activity for L-PGDS gene expression. Together, these results show that PU.1, activated by CKII or NIK, cooperates with MAPK-activated cJun to maximally induce L-PGDS expression in macrophages following LPS treatment, and suggest that PU.1 participates in innate immunity through the production of L-PGDS and PGD(2).

Neurotrimin is an estrogen-regulated determinant of peripheral sympathetic innervation.

Mechanisms underlying axon degeneration in peripheral neuropathies and during normal remodeling are poorly understood. Because estrogen induces widespread sympathetic axon degeneration in female reproductive tract smooth muscle, we surveyed estrogen-regulated genes in rat myometrium. Microarray analysis revealed that the neural cell adhesion protein neurotrimin (Ntm) was markedly up-regulated 6 hr and down-regulated 24 hr after injection of 17beta-estradiol, and real time RT-PCR confirmed this pattern of expression. Protein analysis by Western blotting showed that uterine Ntm protein is also up-regulated in vivo 6-24 hr following estrogen injection and that Ntm protein is increased selectively in the myometrium during the high-estrogen phase of the estrous cycle. Cultured myometrial smooth muscle cells display perinuclear accumulations of Ntm protein, and 17beta-estradiol also increases intracellular levels of Ntm and its secretion into the culture medium. To determine if neurotrimin is required for estrogen-induced sympathetic pruning, sympathetic neurons were cocultured with uterine smooth muscle cells transfected with siRNA directed against Ntm. Although estrogen inhibited neurite outgrowth in nontransfected cocultures, estrogen's ability to reduce sympathetic outgrowth was impaired substantially following Ntm down-regulation. This supports a role for neurotrimin in mediating estrogen-induced sympathetic pruning in some peripheral targets. Together with earlier studies, these findings support the idea that physiological sympathetic axon degeneration is a multifactorial process requiring dynamic regulation of multiple repellant proteins.

Peroxisome proliferator-activated receptor beta/delta expression and activation in lung cancer.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is a ligand-binding inducible transcriptional factor linked to carcinogenesis. Important functions of PPARbeta/delta were demonstrated in series of human epithelial cancers; however, its role in lung cancer remains controversial. We investigated the differential expression level and localization of PPARbeta/delta in tumors and adjacent normal lung tissue, and the effect of PPARbeta/delta activation on lung cancer cell proliferation and apoptosis. PPARbeta/delta was expressed in all studied human non-small cell lung cancers, and strong PPARbeta/delta immunoreactivity was observed in epithelial cells of more than 75% of studied lung tumors. PPARbeta/delta expression was consistently limited to the cancer cells in tumor tissue, while in adjacent normal lung tissue it was limited predominantly to the mononuclear cells. We found that ligand-binding activation of PPARbeta/delta stimulates cell proliferation (an effect that was blocked by a dominant-negative construct of PPARbeta/delta), stimulates anchorage-independent cell growth, and inhibits apoptosis in lung cancer cell lines. Importantly, the activation of PPARbeta/delta induces Akt phosphorylation correlated with up-regulation of PDK1, down-regulation of PTEN, and increased expression of Bcl-xL and COX-2. These findings indicate that PPARbeta/delta exerts proliferative and anti-apoptotic effects via PI3K/Akt1 and COX-2 pathways. In conclusion, PPARbeta/delta is strongly expressed in the majority of lung cancers, and its activation induces proliferative and survival response in non-small cell lung cancer.

NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKKalpha.

The exact physiological role of NF-kappaB-inducing kinase (NIK) in the NF-kappaB activation pathway has not been defined, although it is an upstream kinase of IKKalpha. Recent studies have indicated that IKKalpha is a nucleosomal modifier of NF-kappaB signaling. We hypothesized that NIK generates a proximal signal that contributes to IKKalpha modification of nucleosomal structure through phosphorylation of histone H3 and enhancement of target gene expression. By using a chromatin immunoprecipitation assay, our data show that endogenous IKKalpha is recruited to the promoter site of several NF-kappaB-dependent genes in macrophages. Our data show that immunoreactive NIK is rapidly recruited to nuclear compartment in macrophages in response to treatment with endotoxin where it augments phosphorylation of histone H3 by inducing phosphorylation and kinase activity of IKKalpha. A small interfering RNA knockdown of NIK markedly reduces phosphorylation of histone H3 in endotoxin treated macrophages. These data, together, demonstrate a novel role for NIK as a histone H3 modifier, through an accessory pathway from NIK to IKKalpha, that could play an important role in the endotoxin response through modification of nucleosomal structure.

Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia.

We hypothesized that PU.1 and PU.1 interacting proteins (PIP) binding to the Toll-like receptor 4 (TLR4) promoter is involved in endotoxin-induced upregulation of TLR4 gene expression. Our results employing chromatin immunoprecipitation assays indicate that PU.1 binds to the murine TLR4 promoter both in macrophage cells and, most importantly, in whole lung tissue. Treatment of RAW 264.7 cells with endotoxin induced the association of PU.1 and the TLR4 promoter in a time-dependent manner, and this was closely tied to interactions between the TLR4 promoter and the PIP interferon regulatory factors (IRF)4 and IRF8. PU.1 binding was related to increases in steady-state TLR4 mRNA and total TLR4 protein in RAW cells. Endotoxemia in animals caused the similar inducible interaction between PU.1 and IRF4 and the TLR4 promoter in lung tissue of mice that was treated with a single intraperitoneal injection of endotoxin. PU.1 binding to the TLR4 promoter was not enhanced in the lung tissue of endotoxin-resistant C3H/HeJ mice in response to endotoxemia. Transient transfection studies in RAW cells indicate that inducible binding of PU.1 to the TLR4 promoter is abrogated by a Ser148 to Ala mutation in PU.1. These data suggest that induction of PU.1/PIP binding to the TLR4 promoter is involved in endotoxin response in vivo and may mediate transcriptional changes in TLR4 gene expression.

Regulation of macrophage cyclooxygenase-2 gene expression by modifications of histone H3.

Some transcription factors involved in the regulation of cyclooxygenase 2 (COX-2) expression in macrophage, including NF-kappaB, interact with p300, which contains histone acetyltransferase (HAT) enzyme complex. Chromatin structure is regulated by modifying enzymes, including HAT, and plays an important role in eukaryotic gene regulation through histone modification. We hypothesized that changes in chromatin structure related to phosphorylation and acetylation of histone H3 adjacent to key DNA binding sequence motif in the COX-2 promoter contribute to COX-2 gene activation in macrophages. Sodium butyrate (NaBT) is a short-chain fatty acid that possesses histone deacetyltransferase-inhibiting activity. Our data show that NaBT accentuates LPS-induced COX-2 gene expression at a transcriptional level, even though NaBT alone does not induce the COX-2 gene expression. Using a chromatin immunoprecipitation assay, we showed that costimulation of RAW 264.7 cells with NaBT and LPS synergistically increases COX-2 gene expression through both acetylation and phosphorylation of histone H3 at the promoter site. Our data show that NaBT accentuates LPS-induced COX-2 gene expression through MAP kinase-dependent increase of phosphorylation and acetylation of histone H3 at the COX-2 promoter site. These data indicate that posttranslational modification of histone H3 has a major effect on COX-2 gene expression by macrophages.

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein.

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.

Apoferritin attenuates experimental allergic encephalomyelitis in SJL mice.

Ferritin has been shown to attenuate iron-catalyzed oxidative damage in several experimental conditions. Since oxidative damage has been implicated in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), an animal model of MS, we tested the hypothesis that ferritin would act to attenuate disease. The experimental design was to increase plasma ferritin levels during the active stage of EAE by giving systemic injections of apoferritin and then compare disease activity between these mice and EAE mice administered vehicle. Additional mice received systemic injections of iron, which induces ferritin synthesis, in order to test the effects of exogenous iron on the disease course. Although plasma levels of ferritin were found to be elevated in both apoferritin and iron-injected EAE mice, only apoferritin treatment resulted in a reduction in disease activity compared to EAE mice given vehicle. The suppressive effects of apoferritin administration suggest that the increase in endogenous ferritin levels that have been previously observed in the cerebrospinal fluid of chronic progressive MS patients with active disease might be functioning to limit the severity and spread of tissue damage.