RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein.

Phosphate homeostasis, mediated by dietary intake, renal absorption, and bone deposition, is incompletely understood because of the uncharacterized roles of numerous implicated protein factors. Here, we identified a novel role for one such element, regulator of G protein signaling 14 (RGS14), suggested by genome-wide association studies to associate with dysregulated Pi levels. We show that human RGS14 possesses a carboxy-terminal PDZ ligand required for sodium phosphate cotransporter 2a (NPT2A) and sodium hydrogen exchanger regulatory factor-1 (NHERF1)-mediated renal Pi transport. In addition, we found using isotope uptake measurements combined with bioluminescence resonance energy transfer assays, siRNA knockdown, pull-down and overlay assays, and molecular modeling that secreted proteins parathyroid hormone (PTH) and fibroblast growth factor 23 inhibited Pi uptake by inducing dissociation of the NPT2A-NHERF1 complex. PTH failed to affect Pi transport in cells expressing RGS14, suggesting that it suppresses hormone-sensitive but not basal Pi uptake. Interestingly, RGS14 did not affect PTH-directed G protein activation or cAMP formation, implying a postreceptor site of action. Further pull-down experiments and direct binding assays indicated that NPT2A and RGS14 bind distinct PDZ domains on NHERF1. We showed that RGS14 expression in human renal proximal tubule epithelial cells blocked the effects of PTH and fibroblast growth factor 23 and stabilized the NPT2A-NHERF1 complex. In contrast, RGS14 genetic variants bearing mutations in the PDZ ligand disrupted RGS14 binding to NHERF1 and subsequent PTH-sensitive Pi transport. In conclusion, these findings identify RGS14 as a novel regulator of hormone-sensitive Pi transport. The results suggest that changes in RGS14 function or abundance may contribute to the hormone resistance and hyperphosphatemia observed in kidney diseases.

Inhibition of ezrin causes PKCα-mediated internalization of erbb2/HER2 tyrosine kinase in breast cancer cells.

Unlike other ErbB family members, HER2 levels are maintained on the cell surface when the receptor is activated, allowing prolonged signaling and contributing to its transforming ability. Interactions between HER2, HSP90, PMCA2, and NHERF1 within specialized plasma membrane domains contribute to the membrane retention of HER2. We hypothesized that the scaffolding protein ezrin, which has been shown to interact with NHERF1, might also help stabilize the HER2-PMCA2-NHERF1 complex at the plasma membrane. Therefore, we examined ezrin expression and its relationship with HER2, NHERF1, and PMCA2 levels in murine and human breast cancers. We also used genetic knockdown and/or pharmacologic inhibition of ezrin, HSP90, NHERF1, PMCA2, and HER2 to examine the functional relationships between these factors and membrane retention of HER2. We found ezrin to be expressed at low levels at the apical surface of normal mammary epithelial cells, but its expression is up-regulated and correlates with HER2 expression in hyperplasia and tumors in murine mammary tumor virus-Neu mice, in human HER2-positive breast cancer cell lines, and in ductal carcinoma in situ and invasive breast cancers from human patients. In breast cancer cells, ezrin co-localizes and interacts with HER2, NHERF1, PMCA2, and HSP90 in specialized membrane domains, and inhibiting ezrin disrupts interactions between HER2, PMCA2, NHERF1, and HSP90, inhibiting HER2 signaling and causing PKCα-mediated internalization and degradation of HER2. Inhibition of ezrin synergizes with lapatinib in a PKCα-dependent fashion to inhibit proliferation and promote apoptosis in HER2-positive breast cancer cells. We conclude that ezrin stabilizes a multiprotein complex that maintains active HER2 at the cell surface.

Aspects in controlled drug delivery for topical applications in veterinary medicine.

The controlled release of drugs is an appealing area of research as it provides numerous benefits in veterinary and human medicine. In this paper we attempt to analyze certain aspects related to topical drug delivery systems, their successes and failures, and their place in veterinary medicine. Some emphasis is given to the pharmaceutical aspects of the delivery systems, where the material available made it possible. Purely topical devices, such as cattle ear tags and various collars, as well as some topically administered bioavailable delivery systems are discussed. Special attention is given to hitherto under-evaluated delivery systems, such as topical varnishes. A carefully selected bibliography aims to lead the reader easily to the facts, without providing overwhelming data of varying quality. We believe that the paper may be of interest to practicing veterinarians as well as to pharmaceutical scientists working or considering practice in the area.

Still stable after all these years: perceptions of sexual harassment in academic contexts.

This research examined perceptions of sexual harassment in academic contexts using data sets collected in 1990 (Time 1) and 2000 (Time 2). We assessed the influence of two individual variables--gender and gender role--and one contextual variable--power of the harasser--on perceptions of harassment. When the harasser was a higher-power individual, participants perceived more vignettes as examples of sexual harassment, viewed female targets more positively, and evaluated male harassers more negatively. Contrary to prediction, participants viewed male harassers less negatively at Time 2 than at Time 1. Despite increased media attention and educational interventions, comparisons to the earlier findings highlight remarkable stability in perceptions of harassment over time.

ACE2 interaction with cytoplasmic PDZ protein enhances SARS-CoV-2 invasion.

SARS-CoV-2 is responsible for the global COVID-19 pandemic. Angiotensin converting enzyme 2 (ACE2) is the membrane-delimited receptor for SARS-CoV-2. Lung, intestine, and kidney, major sites of viral infection, express ACE2 that harbors an intracellular, carboxy-terminal PDZ-recognition motif. These organs prominently express the PDZ protein Na+/H+ exchanger regulatory factor-1 (NHERF1). Here, we report NHERF1 tethers ACE2 and augments SARS-CoV-2 cell entry. ACE2 directly binds both NHERF1 PDZ domains. Disruption of either NHERF1 PDZ core-binding motif or the ACE2 PDZ recognition sequence eliminates interaction. Proximity ligation assays establish that ACE2 and NHERF1 interact at constitutive expression levels in human lung and intestine cells. Ablating ACE2 interaction with NHERF1 accelerated SARS-CoV-2 cell entry. Conversely, elimination of the ACE2 C-terminal PDZ-binding motif decreased ACE2 membrane residence and reduced pseudotyped virus entry. We conclude that the PDZ interaction of ACE2 with NHERF1 facilitates SARS-CoV-2 internalization. ß-Arrestin is likely indispensable, as with G protein-coupled receptors.

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines.

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and alphaES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (alphaES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (alphaES). Each of these isoforms were absent in tissue extracts prepared from RAGE(-/-) mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (alphaES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the alphaES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope.

Preconditioning with high mobility group box 1 (HMGB1) induces lipopolysaccharide (LPS) tolerance.

High mobility group box protein 1 (HMGB1) modulates the innate immune response when present in the extracellular compartment. Receptors for HMGB1 include TLR4, TLR2, and the receptor for advanced glycation end products (RAGE). We tested the hypothesis that extracellular HMGB1 can induce LPS tolerance. HMGB1 dose-response experiments were performed on IFN-gamma-differentiated human monocyte-like THP-1 cells. Treatment with 1 microg/ml HMGB1 18 h before exposure to LPS (1 microg/ml) decreased TNF release, NF-kappaB nuclear DNA-binding activity, phosphorylation, and degradation of IkappaBalpha. Preconditioning with HMGB1 alone and HMGB1 in the presence of polymyxin B decreased LPS-mediated, NF-kappaB-dependent luciferase reporter gene expression. The specificity of HMGB1 in tolerance induction was supported further by showing that boiled HMGB1 failed to induce tolerance, and antibodies against HMGB1 blocked the induction of LPS tolerance. Bone marrow-derived macrophages obtained from C57Bl/6 wild-type mice became LPS-tolerant following HMGB1 exposure ex vivo, but macrophages derived from RAGE-deficient mice failed to develop tolerance and responded normally to LPS. Mice preconditioned with HMGB1 (20 microg) 1 h before LPS injection (10 mg/kg) had lower circulating TNF compared with control mice preconditioned with saline vehicle. Similarly, decreased nuclear DNA binding of hepatic NF-kappaB was observed in mice preconditioned with HMGB1. Taken together, these results suggest that extracellular HMGB1 induces LPS tolerance, and the RAGE receptor is required for this induction.

Para-hydroxyphenylpyruvate inhibits the pro-inflammatory stimulation of macrophage preventing LPS-mediated nitro-oxidative unbalance and immunometabolic shift.

Targeting metabolism is emerging as a promising therapeutic strategy for modulation of the immune response in human diseases. In the presented study we used the lipopolysaccharide (LPS)-mediated activation of RAW 264.7 macrophage-like cell line as a model to investigate changes in the metabolic phenotype and to test the effect of p-hydroxyphenylpyruvate (pHPP) on it. pHPP is an intermediate of the PHE/TYR catabolic pathway, selected as analogue of the ethyl pyruvate (EP), which proved to exhibit antioxidant and anti-inflammatory activities. The results obtained show that LPS-priming of RAW 264.7 cell line to the activated M1 state resulted in up-regulation of the inducible nitric oxide synthase (iNOS) expression and consequently of NO production and in release of the pro-inflammatory cytokine IL-6. All these effects were prevented dose dependently by mM concentrations of pHPP more efficiently than EP. Respirometric and metabolic flux analysis of LPS-treated RAW 264.7 cells unveiled a marked metabolic shift consisting in downregulation of the mitochondrial oxidative phosphorylation and upregulation of aerobic glycolysis respectively. The observed respiratory failure in LPS-treated cells was accompanied with inhibition of the respiratory chain complexes I and IV and enhanced production of reactive oxygen species. Inhibition of the respiratory activity was also observed following incubation of human neonatal fibroblasts (NHDF-neo) with sera from septic patients. pHPP prevented all the observed metabolic alteration caused by LPS on RAW 264.7 or by septic sera on NHDF-neo. Moreover, we provide evidence that pHPP is an efficient reductant of cytochrome c. On the basis of the presented results a working model, linking pathogen-associated molecular patterns (PAMPs)-mediated immune response to mitochondrial oxidative metabolism, is put forward along with suggestions for its therapeutic control.

p-Hydroxyphenylpyruvate, an intermediate of the Phe/Tyr catabolism, improves mitochondrial oxidative metabolism under stressing conditions and prolongs survival in rats subjected to profound hemorrhagic shock.

The aim of this study was to test the effect of a small volume administration of p-hydroxyphenylpyruvate (pHPP) in a rat model of profound hemorrhagic shock and to assess a possible metabolic mechanism of action of the compound. The results obtained show that hemorrhaged rats treated with 2-4% of the estimated blood volume of pHPP survived significantly longer (p<0.001) than rats treated with vehicle. In vitro analysis on cultured EA.hy 926 cells demonstrated that pHPP improved cell growth rate and promoted cell survival under stressing conditions. Moreover, pHPP stimulated mitochondria-related respiration under ATP-synthesizing conditions and exhibited antioxidant activity toward mitochondria-generated reactive oxygen species. The compound effects reported in the in vitro and in vivo analyses were obtained in the same millimolar concentration range. These data disclose pHPP as an efficient energetic substrates-supplier to the mitochondrial respiratory chain as well as an antioxidant supporting the view that the compound warrants further evaluation as a therapeutic agent.

Small interfering RNA mediated Poly (ADP-ribose) Polymerase-1 inhibition upregulates the heat shock response in a murine fibroblast cell line.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a highly conserved multifunctional enzyme, and its catalytic activity is stimulated by DNA breaks. The activation of PARP-1 and subsequent depletion of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) contributes to significant cytotoxicity in inflammation of various etiologies. On the contrary, induction of heat shock response and production of heat shock protein 70 (HSP-70) is a cytoprotective defense mechanism in inflammation. Recent data suggests that PARP-1 modulates the expression of a number of cellular proteins at the transcriptional level. In this study, small interfering RNA (siRNA) mediated PARP-1 knockdown in murine wild-type fibroblasts augmented heat shock response as compared to untreated cells (as evaluated by quantitative analysis of HSP-70 mRNA and HSP-70 protein expression). These events were associated with increased DNA binding of the heat shock factor-1 (HSF-1), the major transcription factor of the heat shock response. Co-immunoprecipitation experiments in nuclear extracts of the wild type cells demonstrated that PARP-1directly interacted with HSF-1. These data demonstrate that, in wild type fibroblasts, PARP-1 plays a pivotal role in modulating the heat shock response both through direct interaction with HSF-1 and poly (ADP-ribosylation).

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice. CONCLUSIONS/SIGNIFICANCE: Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.