Chronic Kidney Disease-associated Lung Injury Is Mediated by Phosphate-induced MAPK/AKT Signaling.

Chronic kidney disease (CKD) is associated with systemic phosphate elevations, called hyperphosphatemia. Translational studies have shown that hyperphosphatemia contributes to CKD-associated inflammation and injury in various tissues, including the kidney, heart, liver, and parathyroid gland. Mechanisms underlying pathologic actions of elevated phosphate on cells are not well understood but seem to involve uptake of phosphate through sodium-phosphate cotransporters and phosphate-induced signaling via fibroblast growth factor receptor (FGFR) 1. Clinical studies indicate CKD patients are more likely to develop inflammatory and restrictive lung diseases, such as fibrotic interstitial lung diseases, and here we aimed to determine whether hyperphosphatemia can cause lung injury. We found that a mouse model of CKD and hyperphosphatemia, induced by an adenine-rich diet, develops lung fibrosis and inflammation. Elevation of systemic phosphate levels by administration of a high-phosphate diet in a mouse model of primary lung inflammation and fibrosis, induced by bleomycin, exacerbated lung injury in the absence of kidney damage. Our in vitro studies identified increases of proinflammatory cytokines in human lung fibroblasts exposed to phosphate elevations. Phosphate activated extracellular signal related kinase (ERK) 1/2 and protein kinase B (PKB/AKT) signaling, and pharmacological inhibition of ERK, AKT, FGFR1, or sodium-phosphate cotransporters prevented phosphate-induced proinflammatory cytokine upregulation. Additionally, inhibition of FGFR1 or sodium-phosphate cotransporters decreased the phosphate-induced activation of ERK and AKT. Our study suggests that phosphate can directly target lung fibroblasts and induce an inflammatory response and that hyperphosphatemia in CKD and non-CKD models contributes to lung injury. Phosphate-lowering strategies might protect from CKD-associated lung injury.

Reduced sialylation of airway mucin impairs mucus transport by altering the biophysical properties of mucin.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.

FGF receptors mediate cellular senescence in the cystic fibrosis airway epithelium.

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF.

Reduced Sialylation of Airway Mucin Impairs Mucus Transport by Altering the Biophysical Properties of Mucin.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. Methods: We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-ß) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. Results: We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-ß1 induced collagen expression/deposition. Conclusion: OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-ß1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-ß1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease hallmarked by aberrant ion transport that results in delayed mucus clearance, chronic infection, and progressive lung function decline. Several animal models have been developed to study the airway anatomy and mucus physiology in CF, but they are costly and difficult to maintain, making them less accessible for many applications. A more available CFTR-/- rat model has been developed and characterized to develop CF airway abnormalities, but consistent dosing of pharmacologic agents and longitudinal evaluation remain a challenge. In this study, we report the development and characterization of a novel ex vivo trachea model that utilizes both wild type (WT) and CFTR-/- rat tracheae cultured on a porcine gelatin matrix. Here we show that the ex vivo tracheae remain viable for weeks, maintain a CF disease phenotype that can be readily quantified, and respond to stimulation of mucus and fluid secretion by cholinergic stimulation. Furthermore, we show that ex vivo tracheae may be used for well-controlled pharmacological treatments, which are difficult to perform on freshly excised trachea or in vivo models with this degree of scrutiny. With improved interrogation possible with a durable trachea, we also established firm evidence of a gland secretion defect in CFTR-/- rat tracheae compared to WT controls. Finally, we demonstrate that the ex vivo tracheae can be used to generate high mucus protein yields for subsequent studies, which are currently limited by in vivo mucus collection techniques. Overall, this study suggests that the ex vivo trachea model is an effective, easy to set up culture model to study airway and mucus physiology.

Fibroblast Growth Factor 23 Signaling Does Not Increase Inflammation from Pseudomonas aeruginosa Infection in the Cystic Fibrosis Bronchial Epithelium.

Background and Objectives: Chronic inflammation due to Pseudomonas aeruginosa (PA) infection in people with cystic fibrosis (CF) remains a concerning issue in the wake of modulator therapy initiation. Given the perpetuating cycle of colonization, infection, chronic inflammation, and recurrent injury to the lung, there are increases in the risk for mortality in the CF population. We have previously shown that fibroblast growth factor (FGF) 23 can exaggerate transforming growth factor (TGF) beta-mediated bronchial inflammation in CF. Our study aims to shed light on whether FGF23 signaling also plays a role in PA infection of the CF bronchial epithelium. Materials and Methods: CF bronchial epithelial cells were pretreated with FGF23 or inhibitors for FGF receptors (FGFR) and then infected with different PA isolates. After infection, immunoblot analyses were performed on these samples to assess the levels of phosphorylated phospholipase C gamma (PLCγ), total PLCγ, phosphorylated extracellular signal-regulated kinase (ERK), and total ERK. Additionally, the expression of FGFRs and interleukins at the transcript level (RT-qPCR), as well as production of interleukin (IL)-6 and IL-8 at the protein level (ELISA) were determined. Results: Although there were decreases in isoform-specific FGFRs with increases in interleukins at the mRNA level as well as phosphorylated PLCγ and the production of IL-8 protein with PA infection, treatment with FGF23 or FGFR blockade did not alter downstream targets such as IL-6 and IL-8. Conclusions: FGF23 signaling does not seem to modulate the PA-mediated inflammatory response of the CF bronchial epithelium.

Sustained Increases in Cardiomyocyte Protein O-Linked ß-N-Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment.

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.

A novel in vitro model to study prolonged Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium.

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

Fibroblast Growth Factor Signaling in Development and Disease.

Fibroblast growth factors (FGFs) and their cognate receptors (FGFRs) are important biological molecules with a wide array of pleiotropic functions [...].

Phosphate induces inflammation and exacerbates injury from cigarette smoke in the bronchial epithelium.

An elevation in serum phosphate-also called hyperphosphatemia-is associated with reduced kidney function in chronic kidney disease (CKD). Reports show CKD patients are more likely to develop lung disease and have poorer kidney function that positively correlates with pulmonary obstruction. However, the underlying mechanisms are not well understood. Here, we report that two murine models of CKD, which both exhibit increased serum levels of phosphate and fibroblast growth factor (FGF) 23, a regulator of phosphate homeostasis, develop concomitant airway inflammation. Our in vitro studies point towards a similar increase of phosphate-induced inflammatory markers in human bronchial epithelial cells. FGF23 stimulation alone does not induce a proinflammatory response in the non-COPD bronchial epithelium and phosphate does not cause endogenous FGF23 release. Upregulation of the phosphate-induced proinflammatory cytokines is accompanied by activation of the extracellular-signal regulated kinase (ERK) pathway. Moreover, the addition of cigarette smoke extract (CSE) during phosphate treatments exacerbates inflammation as well as ERK activation, whereas co-treatment with FGF23 attenuates both the phosphate as well as the combined phosphate- and CS-induced inflammatory response, independent of ERK activation. Together, these data demonstrate a novel pathway that potentially explains pathological kidney-lung crosstalk with phosphate as a key mediator.

The Glycobiology of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease of complex etiology. Cases of PAH that do not receive therapy after diagnosis have a low survival rate. Multiple reports have shown that idiopathic PAH, or IPAH, is associated with metabolic dysregulation including altered bioavailability of nitric oxide (NO) and dysregulated glucose metabolism. Multiple processes such as increased proliferation of pulmonary vascular cells, angiogenesis, apoptotic resistance, and vasoconstriction may be regulated by the metabolic changes demonstrated in PAH. Recent reports have underscored similarities between metabolic abnormalities in cancer and IPAH. In particular, increased glucose uptake and altered glucose utilization have been documented and have been linked to the aforementioned processes. We were the first to report a link between altered glucose metabolism and changes in glycosylation. Subsequent reports have highlighted similar findings, including a potential role for altered metabolism and aberrant glycosylation in IPAH pathogenesis. This review will detail research findings that demonstrate metabolic dysregulation in PAH with an emphasis on glycobiology. Furthermore, this report will illustrate the similarities in the pathobiology of PAH and cancer and highlight the novel findings that researchers have explored in the field.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis. Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model. Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac. Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.

ST6GAL1 and α2-6 Sialylation Regulates IL-6 Expression and Secretion in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

Specific O-GlcNAc modification at Ser-615 modulates eNOS function.

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and devastating disease characterized by vascular smooth muscle and endothelial cell proliferation leading to a narrowing of the vessels in the lung. The increased resistance in the lung and the higher pressures generated result in right heart failure. Nitric Oxide (NO) deficiency is considered a hallmark of IPAH and altered function of endothelial nitric oxide synthase (eNOS), decreases NO production. We recently demonstrated that glucose dysregulation results in augmented protein serine/threonine hydroxyl-linked N-Acetyl-glucosamine (O-GlcNAc) modification in IPAH. In diabetes, dysregulated glucose metabolism has been shown to regulate eNOS function through inhibition of Ser-1177 phosphorylation. However, the link between O-GlcNAc and eNOS function remains unknown. Here we show that increased protein O-GlcNAc occurs on eNOS in PAH and Ser-615 appears to be a novel site of O-GlcNAc modification resulting in reduced eNOS dimerization. Functional characterization of Ser-615 demonstrated the importance of this residue on the regulation of eNOS activity through control of Ser-1177 phosphorylation. Here we demonstrate a previously unidentified regulatory mechanism of eNOS whereby the O-GlcNAc modification of Ser-615 results in reduced eNOS activity and endothelial dysfunction under conditions of glucose dysregulation.

Targeting Aging Pathways in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) has become a global epidemic and is the third leading cause of death worldwide. COPD is characterized by chronic airway inflammation, loss of alveolar-capillary units, and progressive decline in lung function. Major risk factors for COPD are cigarette smoking and aging. COPD-associated pathomechanisms include multiple aging pathways such as telomere attrition, epigenetic alterations, altered nutrient sensing, mitochondrial dysfunction, cell senescence, stem cell exhaustion and chronic inflammation. In this review, we will highlight the current literature that focuses on the role of age and aging-associated signaling pathways as well as their impact on current treatment strategies in the pathogenesis of COPD. Furthermore, we will discuss established and experimental COPD treatments including senolytic and anti-aging therapies and their potential use as novel treatment strategies in COPD.

Fibroblast Growth Factor Receptor 4 Deficiency Mediates Airway Inflammation in the Adult Healthy Lung?

Fibroblast growth factor receptor (FGFR) 4 has been shown to mediate pro-inflammatory signaling in the liver and airway epithelium in chronic obstructive pulmonary disease. In past reports, FGFR4 knockout (Fgfr4 -/- ) mice did not show any lung phenotype developmentally or at birth, unless FGFR3 deficiency was present simultaneously. Therefore, we wanted to know whether the loss of FGFR4 had any effect on the adult murine lung. Our results indicate that adult Fgfr4 -/- mice demonstrate a lung phenotype consisting of widened airway spaces, increased airway inflammation, bronchial obstruction, and right ventricular hypertrophy consistent with emphysema. Despite downregulation of FGF23 serum levels, interleukin (IL) 1ß and IL-6 in the Fgfr4 -/- lung, and abrogation of p38 signaling, primary murine Fgfr4 -/- airway cells showed increased expression of IL-1ß and augmented secretion of IL-6, which correlated with decreased airway surface liquid depth as assessed by micro-optical coherence tomography. These findings were paralleled by increased ERK phosphorylation in Fgfr4 -/- airway cells when compared with their control wild-type cells. Analysis of a murine model with constitutive activation of FGFR4 showed attenuation of pro-inflammatory mediators in the lung and airway epithelium. In conclusion, we are the first to show an inflammatory and obstructive airway phenotype in the adult healthy murine Fgfr4 -/- lung, which might be due to the upregulation of ERK phosphorylation in the Fgfr4 -/- airway epithelium.

Upregulation of Sortilin, a Lysosomal Sorting Receptor, Corresponds with Reduced Bioavailability of Latent TGFß in Mucolipidosis II Cells.

Mucolipidosis II (ML-II) is a lysosomal disease caused by defects in the carbohydrate-dependent sorting of soluble hydrolases to lysosomes. Altered growth factor signaling has been identified as a contributor to the phenotypes associated with ML-II and other lysosomal disorders but an understanding of how these signaling pathways are affected is still emerging. Here, we investigated transforming growth factor beta 1 (TGFß1) signaling in the context of ML-II patient fibroblasts, observing decreased TGFß1 signaling that was accompanied by impaired TGFß1-dependent wound closure. We found increased intracellular latent TGFß1 complexes, caused by reduced secretion and stable localization in detergent-resistant lysosomes. Sortilin, a sorting receptor for hydrolases and TGFß-related cytokines, was upregulated in ML-II fibroblasts as well as GNPTAB-null HeLa cells, suggesting a mechanism for inappropriate lysosomal targeting of TGFß. Co-expression of sortilin and TGFß in HeLa cells resulted in reduced TGFß1 secretion. Elevated sortilin levels correlated with normal levels of cathepsin D in ML-II cells, consistent with a compensatory role for this receptor in lysosomal hydrolase targeting. Collectively, these data support a model whereby sortilin upregulation in cells with lysosomal storage maintains hydrolase sorting but suppresses TGFß1 secretion through increased lysosomal delivery. These findings highlight an unexpected link between impaired lysosomal sorting and altered growth factor bioavailability.

Hydrogen peroxide regulates endothelial surface N-glycoforms to control inflammatory monocyte rolling and adhesion.

Monocyte extravasation through the endothelial layer is a hallmark of atherosclerotic plaque development and is mediated by heavily N-glycosylated surface adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1). N-glycosylation is a key co- and post-translational modification that adds sugar molecules to Asparagine residues of surface and secreted proteins. While it has been suggested that surface and secreted proteins will not be expressed unless fully processed to a complex N-glycoform, emerging data has suggested that multiple N-glycoforms can exist on the cell surface. Previous data from our lab has shown that endothelial inflammation produces multiple N-glycoforms of ICAM-1, and that a hypoglycosylated, or high-mannose (HM), form of ICAM-1 enhances adhesion of pro-inflammatory monocytes associated with more severe atherosclerosis and adverse cardiac events. Despite these findings, little is understood about the regulation of N-glycans during disease. In this study, we focus on the α-mannosidases; an understudied class of enzymes for early N-glycan processing. We show that α-mannosidase activity decreases with TNFα treatment in endothelial cells, and this decrease correlates with HM N-glycan formation on the cell surface. Further, we demonstrate that this inhibition is class-I dependent, and is independent of NF-κB upregulation of ICAM-1. Finally, we show that this inhibition is due in part to hydrogen peroxide (H2O2), generated by Endoplasmic Reticulum oxidoreductase 1-α (ERO1α). These data provide insights into the regulation of surface N-glycans during inflammation and demonstrate a novel role for reactive species in N-glycan biosynthesis.