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.

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.

TRPV4 Mechanotransduction in Fibrosis.

Fibrosis is an irreversible, debilitating condition marked by the excessive production of extracellular matrix and tissue scarring that eventually results in organ failure and disease. Differentiation of fibroblasts to hypersecretory myofibroblasts is the key event in fibrosis. Although both soluble and mechanical factors are implicated in fibroblast differentiation, much of the focus is on TGF-ß signaling, but to date, there are no specific drugs available for the treatment of fibrosis. In this review, we describe the role for TRPV4 mechanotransduction in cardiac and lung fibrosis, and we propose TRPV4 as an alternative therapeutic target for fibrosis.

Transient Receptor Potential Vanilloid channel regulates fibroblast differentiation and airway remodeling by modulating redox signals through NADPH Oxidase 4.

Asthma is characterized by pathological airway remodeling resulting from persistent myofibroblast activation. Although transforming growth factor beta 1 (TGFß1), mechanical signals, and reactive oxygen species (ROS) are implicated in fibroblast differentiation, their integration is still elusive. We identified that Transient Receptor Potential Vanilloid 4 (TRPV4), a mechanosensitive ion channel mediates lung fibroblast (LF) differentiation and D. farinae-induced airway remodeling via a novel TRPV4-NADPH Oxidase 4 (NOX4) interaction. NOX4-mediated ROS production is essential for TGFß1-induced LF differentiation via myocardin-related transcription factor-A (MRTF-A) and plasminogen activator inhibitor 1 (PAI-1). Importantly, TRPV4 inhibition prevented TGFß1-induced NOX4 expression and ROS production. Both TRPV4 and NOX4 are activated by phosphatidylinositol 3-kinase (PI3K) downstream of TGFß1, and signals from both TRPV4 and Rac are necessary for NOX4 upregulation. Notably, NOX4 expression is higher in fibroblasts derived from asthmatic patients (disease human LF; DHLF) in comparison to non-asthmatics (normal human LF; NHLF). Further, NOX4 expression is up-regulated in the lungs of D.farinae-treated wild type mice (WT) relative to saline-treated WT, which was attenuated in TRPV4 knockout (KO) mice. Our findings suggest that TRPV4 integrates TGFß1 and ROS signaling through NOX4 and, TRPV4-NOX4 interaction is amenable to target lung remodeling during asthma.

Cysteinyl leukotriene 2 receptor promotes endothelial permeability, tumor angiogenesis, and metastasis.

Cysteinyl leukotrienes (cys-LTs) are proinflammatory mediators that enhance vascular permeability through distinct receptors (CysLTRs). We found that CysLT2R regulates angiogenesis in isolated mouse endothelial cells (ECs) and in Matrigel implants in WT mice and enhances EC contraction and permeability via the Rho-dependent myosin light chain 2 and vascular endothelial (VE)-cadherin axis. Since solid tumors utilize aberrant angiogenesis for their growth and metastasis and their vessels exhibit vascular hyperpermeability, we hypothesized that CysLT2R, via its actions on the endothelium, might regulate tumor growth. Both tumor growth and metastases of adoptively transferred syngeneic Lewis lung carcinoma (LLC) cells are significantly reduced in CysLT2R-null mice (Cysltr2-/-) compared with WT and CysLT1R-null mice (Cysltr1-/-). In WT recipients of LLC cells, CysLT2R expression is significantly increased in the tumor vasculature, compared with CysLT1R. Further, the tumor vasculature in Cysltr2-/- recipients exhibited significantly improved integrity, as revealed by increased pericyte coverage and decreased leakage of i.v.-administered Texas Red-conjugated dextran. Administration of a selective CysLT2R antagonist significantly reduced LLC tumor volume, vessel density, dextran leakage, and metastases in WT mice, highlighting CysLT2R as a VEGF-independent regulator of the vasculature promoting risk of metastasis. Thus, both genetic and pharmacological findings establish CysLT2R as a gateway for angiogenesis and EC dysregulation in vitro and ex vivo and in an in vivo model with a mouse tumor. Our data suggest CysLT2R as a possible target for intervention.