Mast-cell expressed membrane protein-1 is expressed in classical monocytes and alveolar macrophages in idiopathic pulmonary fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß-dependent manner.

Mast-cell expressed membrane protein-1 (MCEMP1) is higher in patients with idiopathic pulmonary fibrosis (IPF) with an increased risk of death. Here we aimed to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared with controls. MCEMP1 is upregulated by transforming growth factor beta (TGFß) at the mRNA and protein levels in monocytic leukemia THP-1 cells. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis-regulatory elements within the MCEMP1 promoter. We also found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.NEW & NOTEWORTHY MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF, is regulated by TGFß, and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity.

Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFß dependent manner.

Background: Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is higher in Idiopathic Pulmonary Fibrosis (IPF) patients with increased risk of death and poor outcomes. Here we seek to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. Methods: MCEMP1 expression was analyzed by single-cell RNA sequencing, immunofluorescence in Peripheral Blood Mononuclear Cells (PBMC) as well as in lung tissues from IPF patients and controls. Chromatin Immunoprecipitation (ChiP) and Proximity Ligation Assay (PLA) were used to study the transcriptional regulation of MCEMP1 . Transient RNA interference and lentivirus transduction were used to knockdown and knock-in MCEMP1 in THP-1 cells to study chemotaxis, adhesion, and migration. Bulk RNA sequencing was used to identify the mechanisms by which MCEMP1 participates in monocyte function. Active RHO pull-down assay was used to validate bulk RNA sequencing results. Results: We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared to controls. MCEMP1 was upregulated by TGFß at the mRNA and protein levels in THP-1. TGFß-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis -regulatory elements within the MCEMP1 promoter. In terms of its function, we found that MCEMP1 regulates TGFß-mediated monocyte chemotaxis, adhesion, and migration. 400 differentially expressed genes were found to increase after TGFß stimulation of THP-1, further increased in MCEMP1 knock-in cells treated with TGFß and decreased in MCEMP1 knockdown cells treated with TGFß. GO annotation analysis of these genes showed enrichment for positive regulation of RHO GTPase activity and signal transduction. While TGFß enhanced RHO GTPase activity in THP-1 cells, this effect was attenuated following MCEMP1 knockdown. Conclusion: MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF. MCEMP1 is regulated by TGFß and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFß in RHO activity. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.

Monocytes and macrophages: emerging mechanisms and novel therapeutic targets in pulmonary fibrosis.

Pulmonary fibrosis results from a plethora of abnormal pathogenetic events. In idiopathic pulmonary fibrosis (IPF), inhalational, environmental, or occupational exposures in genetically and epigenetically predisposed individuals trigger recurrent cycles of alveolar epithelial cell injury, activation of coagulation pathways, chemoattraction, and differentiation of monocytes into monocyte-derived alveolar macrophages (Mo-AMs). When these events happen intermittently and repeatedly throughout the individual's life cycle, the wound repair process becomes aberrant leading to bronchiolization of distal air spaces, fibroblast accumulation, extracellular matrix deposition, and loss of the alveolar-capillary architecture. The role of immune dysregulation in IPF pathogenesis and progression has been underscored in the past mainly after the disappointing results of immunosuppressant use in IPF patients; however, recent reports highlighting the prognostic and mechanistic roles of monocytes and Mo-AMs revived the interest in immune dysregulation in IPF. In this review, we will discuss the role of these cells in the onset and progression of IPF, as well as potential targeted therapies.

High Endothelial Venules Accelerate Naive T Cell Recruitment by Tumor Necrosis Factor-Mediated R-Ras Upregulation.

Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.

Prostaglandin E2 breaks down pericyte-endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras.

A close association between pericytes and endothelial cells (ECs) is crucial to the stability and function of capillary blood vessels and microvessels. The loss or dysfunction of pericytes results in significant disruption of these blood vessels as observed in pathological conditions, including cancer, diabetes, stroke, and Alzheimer's disease. Prostaglandin E2 (PGE2) is a lipid mediator of inflammation, and its tissue concentration is elevated in cancer and neurological disorders. Here, we show that the exposure to PGE2 switches pericytes to a fast-migrating, loosely adhered phenotype that fails to intimately interact with ECs. N-cadherin and connexin-43 in adherens junction and gap junction between pericytes and ECs are downregulated by EP-4 and EP-1-dependent mechanisms, leading to breakdown of the pericyte-EC interaction. Furthermore, R-Ras, a small GTPase important for vascular normalization and vessel stability, is transcriptionally repressed by PGE2 in an EP4-dependent manner. Mouse dermal capillary vessels lose pericyte coverage substantially upon PGE2 injection into the skin. Our results suggest that EP-mediated direct disruption of pericytes by PGE2 is a key process for vascular destabilization. Restoring pericyte-EC interaction using inhibitors of PGE2 signaling may offer a therapeutic strategy in cancer and neurological disorders, in which pericyte dysfunction contributes to the disease progression.

Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element-binding protein (CREB) 3-dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.-Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cyclic AMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

GLI2 cooperates with ZEB1 for transcriptional repression of CDH1 expression in human melanoma cells.

In melanoma cells, high expression of the transcription factor GLI2 is associated with increased invasive potential and loss of E-cadherin expression, an event reminiscent of the epithelial-to-mesenchymal transition (EMT). Herein, we provide evidence that GLI2 represses E-cadherin gene (CDH1) expression in melanoma cells via distinct mechanisms, enhancing transcription of the EMT-activator ZEB1 and cooperative repression of CDH1 gene transcription via direct binding of both GLI2 and ZEB1 to two closely positioned Kruppel-like factor-binding sites within the CDH1 promoter. GLI2 silencing rescued CDH1 expression except in melanoma cell lines in which the CDH1 promoter was hypermethylated. Proximity ligation assays identified GLI2-ZEB1 complexes in melanoma cell nuclei, proportional to endogenous GLI2 and ZEB1 expression, and whose accumulation was enhanced by the classical EMT inducer TGF-ß. These data identify GLI2 as a critical modulator of the cadherin switch in melanoma, a molecular process that is critical for metastatic spread of the disease.

Overlapping activities of TGF-ß and Hedgehog signaling in cancer: therapeutic targets for cancer treatment.

Recent advances in the field of cancer therapeutics come from the development of drugs that specifically recognize validated oncogenic or pro-metastatic targets. The latter may be mutated proteins with altered function, such as kinases that become constitutively active, or critical components of growth factor signaling pathways, whose deregulation leads to aberrant malignant cell proliferation and dissemination to metastatic sites. We herein focus on the description of the overlapping activities of two important developmental pathways often exacerbated in cancer, namely Transforming Growth Factor-ß (TGF-ß) and Hedgehog (HH) signaling, with a special emphasis on the unifying oncogenic role played by GLI1/2 transcription factors. The latter are the main effectors of the canonical HH pathway, yet are direct target genes of TGF-ß/SMAD signal transduction. While tumor-suppressor in healthy and pre-malignant tissues, TGF-ß is often expressed at high levels in tumors and contributes to tumor growth, escape from immune surveillance, invasion and metastasis. HH signaling regulates cell proliferation, differentiation and apoptosis, and aberrant HH signaling is found in a variety of cancers. We discuss the current knowledge on HH and TGF-ß implication in cancer including cancer stem cell biology, as well as the current state, both successes and failures, of targeted therapeutics aimed at blocking either of these pathways in the pre-clinical and clinical settings.