GPR56 Regulates VEGF Production and Angiogenesis during Melanoma Progression

2012 February 15Angiogenesis is a critical step during cancer progression. The VEGF is a major stimulator for angiogenesis and is predominantly contributed by cancer cells in tumors. Inhibition of the VEGF signaling pathway has shown promising therapeutic benefits for cancer patients, but adaptive tumor responses are often observed, indicating the need for further understanding of VEGF regulation. We report that a novel G protein–coupled receptor, GPR56, inhibits VEGF production from the melanoma cell lines and impedes melanoma angiogenesis and growth, through the serine threonine proline-rich segment in its N-terminus and a signaling pathway involving protein kinase Cα. We also present evidence that the two fragments of GPR56, which are generated by autocatalyzed cleavage, played distinct roles in regulating VEGF production and melanoma progression. Finally, consistent with its suppressive roles in melanoma progression, the expression levels of GPR56 are inversely correlated with the malignancy of melanomas in human subjects. We propose that components of the GPR56-mediated signaling pathway may serve as new targets for antiangiogenic treatment of melanoma. Cancer Res; 71(16); 5558–68.National Institutes of Health (U.S.) (U54CA126515)Howard Hughes Medical Institut

Evolution of Mouse Hepatitis Virus (MHV) during Chronic Infection: Quasispecies Nature of the Persisting MHV RNA

AbstractCoronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27Kip1 with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27Kip1 was due not only to increased protein half-life, but also to increased mRNA transcription

Therapeutic Targeting of NF-κB in Acute Lung Injury: A Double-Edged Sword

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a devastating disease that can be caused by a variety of conditions including pneumonia, sepsis, trauma, and most recently, COVID-19. Although our understanding of the mechanisms of ALI/ARDS pathogenesis and resolution has considerably increased in recent years, the mortality rate remains unacceptably high (~40%), primarily due to the lack of effective therapies for ALI/ARDS. Dysregulated inflammation, as characterized by massive infiltration of polymorphonuclear leukocytes (PMNs) into the airspace and the associated damage of the capillary-alveolar barrier leading to pulmonary edema and hypoxemia, is a major hallmark of ALI/ARDS. Endothelial cells (ECs), the inner lining of blood vessels, are important cellular orchestrators of PMN infiltration in the lung. Nuclear factor-kappa B (NF-κB) plays an essential role in rendering the endothelium permissive for PMN adhesion and transmigration to reach the inflammatory site. Thus, targeting NF-κB in the endothelium provides an attractive approach to mitigate PMN-mediated vascular injury, not only in ALI/ARDS, but in other inflammatory diseases as well in which EC dysfunction is a major pathogenic mechanism. This review discusses the role and regulation of NF-κB in the context of EC inflammation and evaluates the potential and problems of targeting it as a therapy for ALI/ARDS

SubAB potentiates actin filament formation and regulates thrombin-induced endothelial permeability.

<p>HPAEC were left untreated <i>(</i><b><i>A</i></b><i>)</i> or <i>(</i><b><i>B</i></b><i>)</i> treated with 0.1 µg/ml of SubAB for 6 hours. The cells were then fixed, permeabilized, and stained with Alexa Fluor 488 labeled phalloidin to visualize the actin filaments. Images were analyzed by Fluorescence microscopy. Results are representative of three experiments. <i>(</i><b><i>C</i></b><i>)</i> HPAEC treated with 0.1 µg/ml of SubAB or mutant SubA_A272B for 6 h were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 3–6 for each condition). ^##<i>p</i><0.01 difference from controls; **<i>p</i><0.01 difference from thrombin stimulated controls. <i>(</i><b><i>D</i></b><i>)</i> Following permeability testing the endothelial monolayer representing various experimental conditions (as indicated in the Figure) was stained for bright field imaging.</p