Prostaglandin E2 attenuates lung fibroblast differentiation via inactivation of yes-associated protein signaling.

Prostaglandin E2 (PGE2 ) has been implicated in counteracting fibroblast differentiation by TGFß1 during pulmonary fibrosis. However, the precise mechanism is not well understood. We show here that PGE2 via EP2 R and EP4 R inhibits the expression of mechanosensory molecules Lysyl Oxidase Like 2 (LOXL2), myocardin-related transcription factor A (MRTF-A), ECM proteins, plasminogen activation inhibitor 1 (PAI-1), fibronectin (FN), α-smooth muscle actin (α-SMA), and redox sensor (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4)) required for TGFß1-mediated fibroblast differentiation. We further demonstrate that PGE2 inhibits fibrotic signaling via Yes-associated protein (YAP) but does so independently from its actions on SMAD phosphorylation and conserved cylindromatosis (CYLD; deubiquitinase) expression. Mechanistically, PGE2 phosphorylates/inactivates YAP downstream of EP2 R/Gαs and restrains its translocation to the nucleus, thus inhibiting its interaction with TEA domain family members (TEADs) and transcription of fibrotic genes. Importantly, pharmacological or siRNA-mediated inhibition of YAP significantly downregulates TGFß1-mediated fibrotic gene expression and myofibroblast formation. Notably, YAP expression is upregulated in the lungs of D. farinae-treated wild type (WT) mice relative to saline-treated WT mice. Our results unravel a unique role for PGE2 -YAP interactions in fibroblast differentiation, and that PGE2 /YAP inhibition can be used as a novel therapeutic target in the treatment of pathological conditions associated with myofibroblasts like asthma.

Epigenetic histone modification by butyrate downregulates KIT and attenuates mast cell function.

Short-chain fatty acid butyrate is produced from the bacterial fermentation of indigestible fiber in the intestinal lumen, and it has been shown to attenuate lung inflammation in murine asthma models. Mast cells (MCs) are initiators of inflammatory response to allergens, and they play an important role in asthma. MC survival and proliferation is regulated by its growth factor stem cell factor (SCF), which acts through the receptor, KIT. It has previously been shown that butyrate attenuates the activation of MCs by allergen stimulation. However, how butyrate mechanistically influences SCF signalling to impact MC function remains unknown. Here, we report that butyrate treatment triggered the modification of MC histones via butyrylation and acetylation, and inhibition of histone deacetylase (HDAC) activity. Further, butyrate treatment caused downregulation of SCF receptor KIT and associated phosphorylation, leading to significant attenuation of SCF-mediated MC proliferation, and pro-inflammatory cytokine secretion. Mechanistically, butyrate inhibited MC function by suppressing KIT and downstream p38 and Erk phosphorylation, and it mediated these effects via modification of histones, acting as an HDAC inhibitor and not via its traditional GPR41 (FFAR3) or GPR43 (FFAR2) butyrate receptors. In agreement, the pharmacological inhibition of Class I HDAC (HDAC1/3) mirrored butyrate's effects, suggesting that butyrate impacts MC function by HDAC1/3 inhibition. Taken together, butyrate epigenetically modifies histones and downregulates the SCF/KIT/p38/Erk signalling axis, leading to the attenuation of MC function, validating its ability to suppress MC-mediated inflammation. Therefore, butyrate supplementations could offer a potential treatment strategy for allergy and asthma via epigenetic alterations in MCs.

Protein Kinase C α and ß compensate for each other to promote stem cell factor-mediated KIT phosphorylation, mast cell viability and proliferation.

Mast cells (MCs) develop from hematopoietic progenitors and differentiate into mature MCs that reside within connective or mucosal tissues. Though the number of MCs in tissues usually remains constant, inflammation and asthma disturb this homeostasis, leading to proliferation of MCs. Understanding the signaling events behind this proliferative response could lead to the development of novel strategies for better management of allergic diseases. MC survival, proliferation, differentiation, and migration are all maintained by a MC growth factor, stem cell factor (SCF) via its receptor, KIT. Here, we explored how protein kinase C (PKC) redundancy influences MC proliferation in bone marrow-derived MC (BMMC). We found that SCF activates PKCα and PKCß isoforms, which in turn modulates KIT phosphorylation and internalization. Further, PKCα and PKCß activate p38 mitogen activated protein kinase (MAPK), and this axis subsequently regulates SCF-induced MC cell proliferation. To ascertain the individual roles of PKCα and PKCß, we knocked down either PKCα or PKCß or both via short hairpin RNA (shRNA) and analyzed KIT phosphorylation, p38 MAPK phosphorylation, and MC viability and proliferation. To our surprise, downregulation of neither PKCα nor PKCß affected MC viability and proliferation. In contrast, blocking both PKCα and PKCß significantly attenuated SCF-induced cell viability and proliferation, suggesting that PKCα and PKCß compensate for each other downstream of SCF signaling to enhance MC viability and proliferation. Our results not only suggest that PKC classical isoforms are novel therapeutic targets for SCF/MC-mediated inflammatory and allergic diseases, but they also emphasize the importance of inhibiting both PKCα and ß isoforms simultaneously to prevent MC proliferation.

Therapeutic Targeting of Stromal-Tumor HGF-MET Signaling in an Organotypic Triple-Negative Breast Tumor Model.

The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple-negative breast cancer (TNBC) that have a rich stroma and limited targeted therapies. However, lack of preclinical human tumor models for mechanistic understanding of tumor-stromal interactions has been an impediment to identify effective treatments against the TME. To address this need, we developed a three-dimensional organotypic tumor model to study interactions of patient-derived cancer-associated fibroblasts (CAF) with TNBC cells and explore potential therapy targets. We found that CAFs predominantly secreted hepatocyte growth factor (HGF) and activated MET receptor tyrosine kinase in TNBC cells. This tumor-stromal interaction promoted invasiveness, epithelial-to-mesenchymal transition, and activities of multiple oncogenic pathways in TNBC cells. Importantly, we established that TNBC cells become resistant to monotherapy and demonstrated a design-driven approach to select drug combinations that effectively inhibit prometastatic functions of TNBC cells. Our study also showed that HGF from lung fibroblasts promotes colony formation by TNBC cells, suggesting that blocking HGF-MET signaling potentially could target both primary TNBC tumorigenesis and lung metastasis. Overall, we established the utility of our organotypic tumor model to identify and therapeutically target specific mechanisms of tumor-stromal interactions in TNBC toward the goal of developing targeted therapies against the TME. IMPLICATIONS: Leveraging a state-of-the-art organotypic tumor model, we demonstrated that CAFs-mediated HGF-MET signaling drive tumorigenic activities in TNBC and presents a therapeutic target.

In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell.

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. KEY POINTS: • A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress.

Leukotriene D4 Upregulates Oxidized Low-Density Lipoprotein Receptor 1 and CD36 to Enhance Oxidized LDL Uptake and Phagocytosis in Macrophages Through Cysteinyl Leukotriene Receptor 1.

Endothelial permeability, leukocyte attachment, and unregulated oxidized LDL (oxLDL) uptake by macrophages leading to the formation of foam cells are all vital in the initiation and progression of atherosclerosis. During inflammation, several inflammatory mediators regulate this process through the expression of distinct oxLDL binding cell surface receptors on macrophages. We have previously shown that Leukotriene D4 (LTD4) promotes endothelial dysfunction, increasing endothelial permeability and enhancing TNFα-mediated attachment of monocytes to endothelium, which hints at its possible role in atherosclerosis. Here we analyzed the effect of LTD4 on macrophage function. Macrophages mainly express CysLT1R and flux calcium in response to LTD4. Further, LTD4 potentiates phagocytosis in macrophages as revealed by the uptake of zymosan particles. Notably, LTD4 augmented macrophage phagocytosis and oxLDL uptake which is sensitive to MK-571 [Montelukast (MK)], a CysLT1R-specific antagonist. Mechanistically, LTD4 upregulated two receptors central to foam cell formation, oxidized low-density lipoprotein receptor-1 (OLR1/LOX-1), and CD36 in a time and dose-dependent manner. Finally, LTD4 enhanced the secretion of chemokines MCP-1 and MIP1ß. Our results suggest that LTD4 contributes to atherosclerosis either through driving foam cell formation or recruitment of immune cells or both. CysLT1R antagonists are safely being used in the treatment of asthma, and the findings from the current study suggest that these can be re-purposed for the treatment of atherosclerosis.

Osmoregulatory Role of Betaine and Betaine/γ-Aminobutyric Acid Transporter 1 in Post-Traumatic Syringomyelia.

Syringomyelia (SM) is primarily characterized by the formation of a fluid-filled cyst that forms in the parenchyma of the spinal cord following injury or other pathology. Recent omics studies in animal models have identified dysregulation of solute carriers, channels, transporters, and small molecules associated with osmolyte regulation during syrinx formation/expansion in the spinal cord. However, their connections to syringomyelia etiology are poorly understood. In this study, the biological functions of the potent osmolyte betaine and its associated solute carrier betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) were studied in SM. First, a rat post-traumatic SM model was used to demonstrate that the BGT1 was primarily expressed in astrocytes in the vicinity of syrinxes. In an in vitro system, we found that astrocytes uptake betaine through BGT1 to regulate cell size under hypertonic conditions. Treatment with BGT1 inhibitors, especially NNC 05-2090, demonstrated midhigh micromolar range potency in vitro that reversed the osmoprotective effects of betaine. Finally, the specificity of these BGT1 inhibitors in the CNS was demonstrated in vivo, suggesting feasibility for targeting betaine transport in SM. In summary, these data provide an enhanced understanding of the role of betaine and its associated solute carrier BGT1 in cell osmoregulation and implicates the active role of betaine and BGT1 in syringomyelia progression.

Fluorescence-based analysis of the intracytoplasmic membranes of type I methanotrophs.

Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells. This fluorescence method is sensitive enough to show not only the characteristic shift in intracytoplasmic membrane formation that is present when methanotrophs are grown with or without copper, but also differences in intracytoplasmic membrane levels at intermediate copper concentrations. This technique can also be employed to monitor dynamic intracytoplasmic membrane changes in the same cell in real time under changing growth conditions. We anticipate that this approach will be of use to researchers wishing to visualize intracytoplasmic membranes who may not have access to electron microscopes. It will also have the capability to relate membrane changes in individual living cells to other measurements by fluorescence labelling or other single-cell analysis methods.