MEK1/2 Inhibition Synergistically Enhances the Preventive Effects of Normobaric Oxygen on Spinal Cord Injury in Decompression Sickness Rats

A previous study from our team found that hyperbaric oxygen (HBO) pretreatment attenuated decompression sickness (DCS) spinal cord injury by upregulating heat shock protein 32 (HSP32) via the ROS/p38 MAPK pathway. Meanwhile, a MEK1/2-negative regulatory pathway was also activated to inhibit HSP32 overexpression. The purpose of this study was to determine if normobaric oxygen (NBO) might effectively induce HSP32 while concurrently inhibiting MEK1/2 and to observe any protective effects on spinal cord injury in DCS rats. The expression of HSP32 in spinal cord tissue was measured at 6, 12, 18, and 24 h following NBO and MEK1/2 inhibitor U0126 pretreatment. The peak time of HSP32 was observed at 12 h after simulated air diving. Subsequently, signs of DCS, hindlimb motor function, and spinal cord and serum injury biomarkers were recorded. NBO-U0126 pretreatment significantly decreased the incidence of DCS, improved motor function, and attenuated oxidative stress, inflammatory response, and apoptosis in both the spinal cord and serum. These results suggest that pretreatment with NBO and U0126 combined can effectively alleviate DCS spinal cord injury in rats by upregulating HSP32. This may lead to a more convenient approach for DCS injury control, using non-pressurized NBO instead of HBO.

Pirh2, an E3 ligase, regulates the AIP4–p73 regulatory pathway by modulating AIP4 expression and ubiquitination

Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

Streptococcus gordoniiprograms epithelial cells to resist ZEB2 induction byPorphyromonas gingivalis

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here thatPorphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial–mesenchymal transition and inflammatory responses. ZEB2 regulation byP. gingivaliswas mediated through pathways involving β-catenin and FOXO1. Among the community partners ofP. gingivalis,Streptococcus gordoniiwas capable of antagonizing ZEB2 expression. Mechanistically,S. gordoniisuppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence ofP. gingivalis. Collectively, these results establishS. gordoniias homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K553 and K573). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

IL-31–IL-31R interactions negatively regulate type 2 inflammation in the lung

Interleukin (IL) 31Rα (glycoprotein 130–like monocyte receptor and glycoprotein 130–like receptor) heterodimerizes with oncostatin M receptor β to bind IL-31, a cytokine expressed preferentially by CD4+ T helper type 2 (Th2) cells. However, the functions of IL-31–IL-31R signaling in immune regulation remain unknown. Here, we identify a novel role for IL-31R in limiting type 2 inflammation in the lung. After intravenous injection of Schistosoma mansoni eggs, IL-31Rα−/− mice developed severe pulmonary inflammation, characterized by an increase in the area of granulomatous inflammation, increased numbers of resistin-like molecule α+ cells, and enhanced collagen deposition compared to WT counterparts. In vitro, macrophages generated from IL-31Rα−/− mice promoted enhanced ovalbumin-specific CD4+ T cell proliferation and purified naive CD4+ T cells from IL-31Rα−/− mice exhibited enhanced proliferation and expression of Th2 cytokines, identifying a T cell– and macrophage-intrinsic regulatory function for IL-31R signaling. In contrast, the generation of CD4+ T cell–mediated Th1 responses were normal in IL-31Rα−/− mice, suggesting that the regulatory role of IL-31R signaling is limited to type 2 responses. Together, these data implicate IL-31R signaling as a novel negative regulatory pathway that specifically limits type 2 inflammation.

The protein kinase C inhibitor RO318220 potentiates thrombin-stimulated platelet-supported prothrombinase activity

Prothrombinase activity was tested on thrombin- and SFLLRN-activated platelets treated with RO318220, a potent inhibitor of protein kinase C. RO318220 completely inhibited platelet dense and α-granule secretion at a concentration of 20 μM but had no effect on prothrombinase activity in the presence of excess factor Va (20 nM). This indicates that protein kinase C activity and agonist-initiated secretion are not necessary for the development of a procoagulant surface. Treatment with 75 to 150 μM RO318220 potentiated platelet-supported thrombin generation up to 280% of control platelets with no change in Kd appFXa. Treated with increasing concentrations of RO318220, an increasing proportion of thrombin-stimulated platelets bound annexin V with decreasing binding sites per platelet. A lower mean forward scatter (FSC-H) of platelets treated with RO318220 suggested platelet vesiculation as a result of RO318220 treatment; however, 100 μM calpeptin pretreatment eliminated the decrease in FSC-H without affecting either the increase in platelets positive for annexin V binding, the decrease in binding sites per platelet, or the 3-fold increase in prothrombinase activity. Thus, RO318220 appears to increase prothrombinase activity by increasing platelet responsiveness to thrombin rather than by inducing platelet vesiculation. This suggests that RO318220 inhibits a signaling molecule within a negative regulatory pathway that governs platelet procoagulant surface changes. (Blood. 2003;102:2472-2481)

Hematopoietic progenitor kinase 1 (HPK1) negatively regulates prostaglandin E2–induced fos gene transcription

Prostaglandin E2 (PGE2) is the predominant eicosanoid product released by macrophages at the site of inflammation. Binding of PGE2 to its cognate 7 transmembrane-spanning G protein–coupled receptors (GPCRs) activates signaling pathways, leading to the synthesis of the Fos transcription factor. Because the Ste20 serine/threonine protein kinase (S/TPK) is a critical signal transducer for the G protein–coupled pheromone receptor in Saccharomyces cerevisiae, we postulated that the PGE2 GPCRs may activate one of the Ste20 mammalian orthologs. We demonstrate here that the catalytic activity of a hematopoietic cell–restricted, Ste20-related S/TPK, HPK1, is positively regulated by exposure to physiological concentrations of PGE2. Furthermore, ectopic expression studies implicated HPK1 as a negative regulator of PGE2-induced transcription of the fos gene. Our data suggest that PGE2-induced activation of HPK1 may represent a novel negative regulatory pathway capable of modulating PGE2-mediated gene transcription.

Isolation and Characterization of the cDNA for Mouse Neutrophil Collagenase: Demonstration of Shared Negative Regulatory Pathways for Neutrophil Secondary Granule Protein Gene Expression

A characteristic of normal neutrophil maturation is the induction of secondary granule protein (SGP) mRNA expression. Several leukemic human cell lines mimic normal morphologic neutrophil differentiation but fail to express SGPs, such as lactoferrin (LF) and neutrophil gelatinase (NG). In contrast, two murine cell lines (32D C13 and MPRO) are able to differentiate into neutrophils and induce expression of LF and NG. Therefore, to study the normal regulation and function of these genes, the corresponding murine homologs must be isolated. Using cDNA representational difference analysis (RDA) to compare a committed myeloid progenitor cell line (EPRO) with the multipotent stem cell line from which it was derived (EML), we isolated a fragment bearing homology to human neutrophil collagenase (hNC). Here, we describe the cloning and characterization of a full-length (∼2 kb) clone that exhibits nearly 65% nucleotide and 73% amino acid identity to hNC. Ribonuclease protection analysis (RPA) of the tissues and cell lines shows that mouse NC (mNC) is expressed only in cell lines exhibiting neutrophilic characteristics, further confirming its identity as the mouse homolog of hNC. Furthermore, we have demonstrated a shared negative regulatory pathway for this and other SGP genes. We have previously shown that CCAAT displacement protein (CDP/cut) binds to a specific region of the LF promoter, and overexpression of CDP blocks G-CSF–induced upregulation of LF gene expression in 32D C13 cells. We show here that in these cells, upregulation of both NC and NG is also blocked. CDP is thus the first identified transcription factor that is a candidate for mediating the shared regulation of neutrophil SGP protein genes.