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1.
Physiol Rev ; 100(1): 463-488, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31539306

ABSTRACT

Molecular oxygen (O2) and carbon dioxide (CO2) are the primary gaseous substrate and product of oxidative phosphorylation in respiring organisms, respectively. Variance in the levels of either of these gasses outside of the physiological range presents a serious threat to cell, tissue, and organism survival. Therefore, it is essential that endogenous levels are monitored and kept at appropriate concentrations to maintain a state of homeostasis. Higher organisms such as mammals have evolved mechanisms to sense O2 and CO2 both in the circulation and in individual cells and elicit appropriate corrective responses to promote adaptation to commonly encountered conditions such as hypoxia and hypercapnia. These can be acute and transient nontranscriptional responses, which typically occur at the level of whole animal physiology or more sustained transcriptional responses, which promote chronic adaptation. In this review, we discuss the mechanisms by which mammals sense changes in O2 and CO2 and elicit adaptive responses to maintain homeostasis. We also discuss crosstalk between these pathways and how they may represent targets for therapeutic intervention in a range of pathological states.


Subject(s)
Carbon Dioxide/metabolism , Homeostasis , Mammals/physiology , Oxygen/metabolism , Acidosis, Respiratory , Animals , Humans , Hypercapnia , Hypocapnia , Hypoxia , Mammals/metabolism
2.
J Immunol ; 208(10): 2363-2375, 2022 05 15.
Article in English | MEDLINE | ID: mdl-35477686

ABSTRACT

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.


Subject(s)
Carbon Dioxide , Carbonic Anhydrase II , Carbon Dioxide/metabolism , Carbonic Anhydrase II/metabolism , Humans , Hydrogen-Ion Concentration , Hypercapnia/enzymology , Hypercapnia/metabolism , Isoenzymes
3.
Int J Mol Sci ; 25(5)2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38474099

ABSTRACT

Hypercapnia occurs when the partial pressure of carbon dioxide (CO2) in the blood exceeds 45 mmHg. Hypercapnia is associated with several lung pathologies and is transcriptionally linked to suppression of immune and inflammatory signalling through poorly understood mechanisms. Here we propose Orphan Nuclear Receptor Family 4A (NR4A) family members NR4A2 and NR4A3 as potential transcriptional regulators of the cellular response to hypercapnia in monocytes. Using a THP-1 monocyte model, we investigated the sensitivity of NR4A family members to CO2 and the impact of depleting NR4A2 and NR4A3 on the monocyte response to buffered hypercapnia (10% CO2) using RNA-sequencing. We observed that NR4A2 and NR4A3 are CO2-sensitive transcription factors and that depletion of NR4A2 and NR4A3 led to reduced CO2-sensitivity of mitochondrial and heat shock protein (Hsp)-related genes, respectively. Several CO2-sensitive genes were, however, refractory to depletion of NR4A2 and NR4A3, indicating that NR4As regulate certain elements of the cellular response to buffered hypercapnia but that other transcription factors also contribute. Bioinformatic analysis of conserved CO2-sensitive genes implicated several novel putative CO2-sensitive transcription factors, of which the ETS Proto-Oncogene 1 Transcription Factor (ETS-1) was validated to show increased nuclear expression in buffered hypercapnia. These data give significant insights into the understanding of immune responses in patients experiencing hypercapnia.


Subject(s)
Orphan Nuclear Receptors , Receptors, Steroid , Humans , Orphan Nuclear Receptors/genetics , Monocytes/metabolism , Hypercapnia , Carbon Dioxide , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Receptors, Steroid/metabolism , DNA-Binding Proteins , Receptors, Thyroid Hormone
4.
Immunol Cell Biol ; 101(6): 556-577, 2023 Jul.
Article in English | MEDLINE | ID: mdl-36967673

ABSTRACT

CO2 is produced during aerobic respiration. Normally, levels of CO2 in the blood are tightly regulated but pCO2 can rise (hypercapnia, pCO2 > 45 mmHg) in patients with lung diseases, for example, chronic obstructive pulmonary disease (COPD). Hypercapnia is a risk factor in COPD but may be of benefit in the context of destructive inflammation. The effects of CO2 per se, on transcription, independent of pH change are poorly understood and warrant further investigation. Here we elucidate the influence of hypercapnia on monocytes and macrophages through integration of state-of-the-art RNA-sequencing, metabolic and metabolomic approaches. THP-1 monocytes and interleukin 4-polarized primary murine macrophages were exposed to 5% CO2 versus 10% CO2 for up to 24 h in pH-buffered conditions. In hypercapnia, we identified around 370 differentially expressed genes (DEGs) under basal and about 1889 DEGs under lipopolysaccharide-stimulated conditions in monocytes. Transcripts relating to both mitochondrial and nuclear-encoded gene expression were enhanced in hypercapnia in basal and lipopolysaccharide-stimulated cells. Mitochondrial DNA content was not enhanced, but acylcarnitine species and genes associated with fatty acid metabolism were increased in hypercapnia. Primary macrophages exposed to hypercapnia also increased activation of genes associated with fatty acid metabolism and reduced activation of genes associated with glycolysis. Thus, hypercapnia elicits metabolic shifts in lipid metabolism in monocytes and macrophages under pH-buffered conditions. These data indicate that CO2 is an important modulator of monocyte transcription that can influence immunometabolic signaling in immune cells in hypercapnia. These immunometabolic insights may be of benefit in the treatment of patients experiencing hypercapnia.


Subject(s)
Hypercapnia , Pulmonary Disease, Chronic Obstructive , Humans , Animals , Mice , Hypercapnia/etiology , Hypercapnia/metabolism , Carbon Dioxide , Monocytes/metabolism , Genes, Mitochondrial , Lipopolysaccharides , Pulmonary Disease, Chronic Obstructive/complications , Gene Expression , Fatty Acids
5.
Biochem Biophys Res Commun ; 555: 19-25, 2021 05 28.
Article in English | MEDLINE | ID: mdl-33812054

ABSTRACT

Adenosine is a purine nucleoside pivotal for homeostasis in cells and tissues. Stimulation of the adenosine receptors (AR) has been shown to regulate the nuclear orphan receptor 4A (NR4A1-3) family, resulting in attenuation of hyper-inflammatory responses in myeloid cells. The NR4A1-3 orphan receptors are early immediate response genes and transcriptional regulators of cell and tissue homeostasis. The signal transduction and transcriptional mechanism(s) of how AR-stimulation promotes NR4A expression in myeloid cells is unknown and is the focus of this study. We confirm that adenosine and the stable analogue, 5'-N-Ethylcarboxamidoadenosine (NECA), enhance NR4A1-3 expression in THP-1 cells. Pharmacological approaches identified that protein kinase D (PKD) mediates AR-stimulated NR4A expression in myeloid cells and reveals no involvement of PKA nor PKC. The role of NF-κB, a principal regulator of NR4A expression in myeloid cells, was examined as a possible transcriptional regulator downstream of PKD. Utilising BAY11-7082 and MG-132, inhibitors of the respective ubiquitin and proteasome pathways essential for NF-κB activation, suggested a prospective role for NF-κB, or more specifically signalling via IKKα/ß. However, biological interventional studies using overexpression of IκBα in myeloid cells and MEF cells lacking IKKα and IKKß (IKKα/ß-/-) revealed the NF-κB pathway is not utilised in mediating AR-stimulated NR4A expression. Thus, this study contributes mechanistic insight into how AR signalling modulates the expression of NR4A receptors, pivotal regulators of inflammatory responses in myeloid cells.


Subject(s)
Myeloid Cells/metabolism , Orphan Nuclear Receptors/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Kinase C/metabolism , Receptors, Purinergic P1/metabolism , Adenosine/administration & dosage , Adenosine/metabolism , Adenosine/pharmacology , Adenosine-5'-(N-ethylcarboxamide)/administration & dosage , Adenosine-5'-(N-ethylcarboxamide)/pharmacology , DNA-Binding Proteins/metabolism , Humans , NF-kappa B/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Receptors, Steroid/metabolism , Receptors, Thyroid Hormone/metabolism , THP-1 Cells , Ubiquitin/metabolism
6.
Biochem Biophys Res Commun ; 554: 179-185, 2021 05 21.
Article in English | MEDLINE | ID: mdl-33798945

ABSTRACT

Inflammation is a pivotal pathological factor in colorectal cancer (CRC) initiation and progression, and modulating this inflammatory state has the potential to ameliorate disease progression. NR4A receptors have emerged as key regulators of inflammatory pathways that are important in CRC. Here, we have examined the effect of NR4A agonist, Cytosporone B (CsnB), on colorectal tissue integrity and its effect on the inflammatory profile in CRC tissue ex vivo. Here, we demonstrate concentrations up 100 µM CsnB did not adversely affect tissue integrity as measured using transepithelial electrical resistance, histology and crypt height. Subsequently, we reveal through the use of a cytokine/chemokine array, ELISA and qRT-PCR analysis that multiple pro-inflammatory mediators were significantly increased in CRC tissue compared to control tissue, which were then attenuated with the addition of CsnB (such as IL-1ß, IL-8 and TNFα). Lastly, stratification of the data revealed that CsnB especially alters the inflammatory profile of tumours derived from males who had not undergone chemoradiotherapy. Thus, this study demonstrates that NR4A agonist CsnB does not adversely affect colon tissue structure or functionality and can attenuate the pro-inflammatory state of human CRC tissue ex vivo.


Subject(s)
Colorectal Neoplasms/drug therapy , Inflammation Mediators/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/agonists , Phenylacetates/pharmacology , Adult , Aged , Aged, 80 and over , Chemokines/metabolism , Colorectal Neoplasms/immunology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cytokines/metabolism , Female , Humans , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Male , Middle Aged
7.
J Immunol ; 202(5): 1521-1530, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30700584

ABSTRACT

Hypoxia is a common and prominent feature of the microenvironment at sites of bacteria-associated inflammation in inflammatory bowel disease. The prolyl-hydroxylases (PHD1/2/3) and the asparaginyl-hydroxylase factor-inhibiting HIF are oxygen-sensing enzymes that regulate adaptive responses to hypoxia through controlling the activity of HIF and NF-κB-dependent transcriptional pathways. Previous studies have demonstrated that the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG) is effective in the alleviation of inflammation in preclinical models of inflammatory bowel disease, at least in part, through suppression of IL-1ß-induced NF-κB activity. TLR-dependent signaling in immune cells, such as monocytes, which is important in bacteria-driven inflammation, shares a signaling pathway with IL-1ß. In studies into the effect of pharmacologic hydroxylase inhibition on TLR-induced inflammation in monocytes, we found that DMOG selectively triggers cell death in cultured THP-1 cells and primary human monocytes at concentrations well tolerated in other cell types. DMOG-induced apoptosis was independent of increased caspase-3/7 activity but was accompanied by reduced expression of the inhibitor of apoptosis protein 1 (cIAP1). Based on these data, we hypothesize that pharmacologic inhibition of the HIF-hydroxylases selectively targets monocytes for cell death and that this may contribute to the anti-inflammatory activity of HIF-hydroxylase inhibitors.


Subject(s)
Amino Acids, Dicarboxylic/pharmacology , Inflammation/drug therapy , Mixed Function Oxygenases/antagonists & inhibitors , Monocytes/drug effects , Prolyl-Hydroxylase Inhibitors/pharmacology , Cell Death/drug effects , Cell Death/immunology , Cells, Cultured , HEK293 Cells , Humans , Inflammation/immunology , Inflammation/metabolism , Mixed Function Oxygenases/immunology , Mixed Function Oxygenases/metabolism , Monocytes/immunology , Monocytes/metabolism
8.
Eur Respir J ; 56(2)2020 08.
Article in English | MEDLINE | ID: mdl-32265303

ABSTRACT

In January 2019, a European Respiratory Society research seminar entitled "Targeting the detrimental effects of sleep disturbances and disorders" was held in Dublin, Ireland. It provided the opportunity to critically review the current evidence of pathophysiological responses of sleep disturbances, such as sleep deprivation, sleep fragmentation or circadian misalignment and of abnormalities in physiological gases such as oxygen and carbon dioxide, which occur frequently in respiratory conditions during sleep. A specific emphasis of the seminar was placed on the evaluation of the current state of knowledge of the pathophysiology of cardiovascular and metabolic diseases in obstructive sleep apnoea (OSA). Identification of the detailed mechanisms of these processes is of major importance to the field and this seminar offered an ideal platform to exchange knowledge, and to discuss pitfalls of current models and the design of future collaborative studies. In addition, we debated the limitations of current treatment strategies for cardiometabolic complications in OSA and discussed potentially valuable alternative approaches.


Subject(s)
Cardiovascular Diseases , Sleep Apnea, Obstructive , Cardiovascular Diseases/therapy , Humans , Ireland , Precision Medicine , Sleep , Sleep Apnea, Obstructive/complications , Sleep Apnea, Obstructive/therapy
9.
PLoS Biol ; 14(1): e1002347, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26752685

ABSTRACT

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.


Subject(s)
Cysteine Endopeptidases/metabolism , Mixed Function Oxygenases/metabolism , Repressor Proteins/metabolism , AMP-Activated Protein Kinases/metabolism , Cysteine Endopeptidases/genetics , Deubiquitinating Enzymes , Energy Metabolism , HEK293 Cells , Humans , Hydroxylation , Mutagenesis, Site-Directed , Protein Stability
10.
Proc Natl Acad Sci U S A ; 113(48): E7778-E7787, 2016 11 29.
Article in English | MEDLINE | ID: mdl-27856732

ABSTRACT

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.


Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Immunity, Innate , Macrophages/metabolism , Pyruvates/metabolism , Trypanosoma brucei brucei/immunology , Trypanosomiasis, African/immunology , Animals , Cell Line , Glycolysis , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immune Evasion , Indoles/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/parasitology , Lipopolysaccharides/pharmacology , Macrophages/parasitology , Mice, Inbred C57BL , Trypanosomiasis, African/parasitology
11.
J Biol Chem ; 292(27): 11561-11571, 2017 07 07.
Article in English | MEDLINE | ID: mdl-28507099

ABSTRACT

CO2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO2 are not fully elucidated. Here, we identify several novel CO2-dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO2 A cohort of RelB protein-protein interactions (e.g. with Raf-1 and IκBα) are altered by CO2 exposure, although others are maintained (e.g. with p100). RelB is processed by CO2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO2 Thus, we provide molecular insight into the CO2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO2-dependent regulation of inflammatory signaling.


Subject(s)
Carbon Dioxide/immunology , Hypercapnia/immunology , NF-kappa B p52 Subunit/immunology , Signal Transduction/immunology , Transcription Factor RelB/immunology , A549 Cells , Animals , Humans , Hypercapnia/genetics , Hypercapnia/pathology , Mice , NF-kappa B p52 Subunit/genetics , Protein Domains , Signal Transduction/genetics , Transcription Factor RelB/genetics , Transcription, Genetic/genetics , Transcription, Genetic/immunology
12.
J Biol Chem ; 291(22): 11800-8, 2016 May 27.
Article in English | MEDLINE | ID: mdl-27044749

ABSTRACT

Molecular oxygen and carbon dioxide are the primary gaseous substrate and product of oxidative metabolism, respectively. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are co-incidental features of the tissue microenvironment in a range of pathophysiologic states, including acute and chronic respiratory diseases. The hypoxia-inducible factor (HIF) is the master regulator of the transcriptional response to hypoxia; however, little is known about the impact of hypercapnia on gene transcription. Because of the relationship between hypoxia and hypercapnia, we investigated the effect of hypercapnia on the HIF pathway. Hypercapnia suppressed HIF-α protein stability and HIF target gene expression both in mice and cultured cells in a manner that was at least in part independent of the canonical O2-dependent HIF degradation pathway. The suppressive effects of hypercapnia on HIF-α protein stability could be mimicked by reducing intracellular pH at a constant level of partial pressure of CO2 Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase that blocks lysosomal degradation, prevented the hypercapnic suppression of HIF-α protein. Based on these results, we hypothesize that hypercapnia counter-regulates activation of the HIF pathway by reducing intracellular pH and promoting lysosomal degradation of HIF-α subunits. Therefore, hypercapnia may play a key role in the pathophysiology of diseases where HIF is implicated.


Subject(s)
Carbon Dioxide/blood , Hypercapnia/physiopathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/physiopathology , Oxygen/metabolism , Animals , Blotting, Western , Cells, Cultured , Female , HCT116 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction
13.
J Immunol ; 195(4): 1436-48, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26150530

ABSTRACT

Adenosine receptor-mediated regulation of monocyte/macrophage inflammatory responses is critical in the maintenance of tissue homeostasis. In this study, we reveal that adenosine potently modulates the expression of NR4A1, 2, and 3 orphan nuclear receptors in myeloid cells, and this modulation is primarily through the adenosine A2a receptor subtype. We demonstrate that A2a receptor activation of NR4A1-3 receptor synthesis is further enhanced in TLR4-stimulated monocytes. After TLR4 stimulation, NR4A receptor-depleted monocyte/macrophage cells display significantly altered expression of cell-surface markers and produce increased inflammatory cytokine and chemokine secretion rendering the cells an enhanced proinflammatory phenotype. Exposure of TLR4 or TNF-α-stimulated monocytes to adenosine analogs directs changes in the expression of MIP-3α and IL-23p19, with NR4A2 depletion leading to significantly enhanced expression of these factors. Furthermore, we establish that nuclear levels of NF-κB/p65 are increased in TLR/adenosine-stimulated NR4A2-depleted cells. We show that, after TLR/adenosine receptor stimulation, NR4A2 depletion promotes significant binding of NF-κB/p65 to a κB consensus binding motif within the MIP-3α proximal promoter leading to increased protein secretion, confirming a pivotal role for NF-κB activity in controlling cellular responses and gene expression outcomes in response to these mediators. Thus, these data demonstrate that during an inflammatory response, adenosine modulation of NR4A receptor activity acts to limit NF-κB-mediated effects and that loss of NR4A2 expression leads to enhanced NF-κB activity and hyperinflammatory responses in myeloid cells.


Subject(s)
Adenosine/metabolism , Monocytes/immunology , Monocytes/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Animals , Cell Line , Chemokine CCL20/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation , Humans , Hypersensitivity/genetics , Hypersensitivity/immunology , Hypersensitivity/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Interleukin-23 Subunit p19/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Models, Biological , NF-kappa B/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 3/genetics , Nuclear Receptor Subfamily 4, Group A, Member 3/metabolism , Orphan Nuclear Receptors , Receptor, Adenosine A2A/metabolism
14.
Am J Physiol Renal Physiol ; 311(1): F35-45, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27122540

ABSTRACT

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3ß (Ser9), nuclear accumulation of ß-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-ß (TGF-ß); Wnt6 reversed TGF-ß-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-ß-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/ß(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.


Subject(s)
Cell Differentiation/genetics , Kidney Diseases/genetics , Kidney Diseases/pathology , Kidney/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Wnt Proteins/genetics , Wnt Proteins/physiology , Animals , Epithelial Cells/pathology , Female , Fibrosis , Frizzled Receptors , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , I-kappa B Proteins/genetics , Kidney/embryology , Kidney Diseases/chemically induced , Kidney Tubules/growth & development , Mice , Mice, Knockout , Phosphorylation , Pregnancy , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Signal Transduction/genetics , Transcription Factor RelA/genetics , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Vimentin/biosynthesis
15.
Am J Physiol Gastrointest Liver Physiol ; 311(6): G1076-G1090, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27789456

ABSTRACT

Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease, a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases, which confer oxygen sensitivity upon the hypoxia-inducible factor pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate, followed by a period of recovery, developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine ameliorated fibrosis. TGF-ß1 is a key regulator of fibrosis that acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-ß1-induced expression of fibrotic markers in cultured fibroblasts, suggesting a direct role for hydroxylases in TGF-ß1 signaling. This was at least in part due to inhibition of noncanonical activation of extracellular signal-regulated kinase (ERK) signaling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis through suppression of TGF-ß1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress profibrotic pathways.


Subject(s)
Collagen/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Intestines/pathology , Mixed Function Oxygenases/metabolism , Transforming Growth Factor beta1/metabolism , Amino Acids, Dicarboxylic/pharmacology , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibrosis , Humans , Intestinal Mucosa/metabolism , Intestines/drug effects , Mice , Mice, Inbred C57BL , Mixed Function Oxygenases/antagonists & inhibitors , Signal Transduction
16.
Biochem Biophys Res Commun ; 474(3): 579-586, 2016 06 03.
Article in English | MEDLINE | ID: mdl-27130823

ABSTRACT

Hepatocyte death is an important contributing factor in a number of diseases of the liver. PHD1 confers hypoxic sensitivity upon transcription factors including the hypoxia inducible factor (HIF) and nuclear factor-kappaB (NF-κB). Reduced PHD1 activity is linked to decreased apoptosis. Here, we investigated the underlying mechanism(s) in hepatocytes. Basal NF-κB activity was elevated in PHD1(-/-) hepatocytes compared to wild type controls. ChIP-seq analysis confirmed enhanced binding of NF-κB to chromatin in regions proximal to the promoters of genes involved in the regulation of apoptosis. Inhibition of NF-κB (but not knock-out of HIF-1 or HIF-2) reversed the anti-apoptotic effects of pharmacologic hydroxylase inhibition. We hypothesize that PHD1 inhibition leads to altered expression of NF-κB-dependent genes resulting in reduced apoptosis. This study provides new information relating to the possible mechanism of therapeutic action of hydroxylase inhibitors that has been reported in pre-clinical models of intestinal and hepatic disease.


Subject(s)
Apoptosis/physiology , Hepatocytes/cytology , Hepatocytes/physiology , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , NF-kappa B/metabolism , Procollagen-Proline Dioxygenase/metabolism , Animals , Cell Hypoxia/physiology , Cell Line , Gene Expression Regulation, Enzymologic/physiology , HEK293 Cells , Humans , Mice
17.
Exp Physiol ; 101(8): 986-1002, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27474261

ABSTRACT

What is the topic of this review? This review highlights the transcriptional consequences for decreased cellular O2 levels (hypoxia) and increased cellular CO2 levels (hypercapnia). What advances does it highlight? We discuss recent advances in our understanding of the cellular response to hypoxia and consider the potential cross-talk between O2 - and CO2 -dependent transcriptional regulation. Oxygen and carbon dioxide are the substrate and product of aerobic metabolism, respectively. Thus, the levels of these physiological gases are inextricably linked in physiological and pathophysiological conditions. Increased mitochondrial consumption of O2 (to produce ATP) will produce more CO2 . Furthermore, in lung pathologies such as chronic obstructive pulmonary disease, sleep apnoea and central hypoventilation syndrome, hypoxia and hypercapnia are co-incident. Acute responses to hypoxia involve carotid body-mediated changes in the rate and depth of breathing. Chronic adaptation to hypoxia involves a multitude of changes on a transcriptional level, which simultaneously increases oxygen utilization (via hypoxia-inducible factor and others), while suppressing superfluous energy-demanding processes. Acute responses to CO2 affect breathing primarily via central chemoreceptors. The nature of hypercapnia-dependent transcriptional regulation is an emerging area of research, but at present the mechanisms underpinning this response are not fully characterized and understood. Thus, given the juxtaposition of hypoxia and hypercapnia in health and disease, this manuscript reviews the current evidence for transcriptional responses to hypoxia and hypercapnia. Finally, we discuss the potential cross-talk between hypoxia and hypercapnia on a transcriptional level.


Subject(s)
Carbon Dioxide/metabolism , Gene Expression Regulation/physiology , Oxygen/metabolism , Transcription, Genetic/physiology , Animals , Humans , Hypercapnia/metabolism , Hypercapnia/physiopathology , Hypoxia/metabolism , Hypoxia/physiopathology
18.
Proc Natl Acad Sci U S A ; 110(46): 18490-5, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24145445

ABSTRACT

Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1ß, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1ß-induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1ß-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1ß signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1ß-dependent inflammatory signaling.


Subject(s)
Gene Expression Regulation/physiology , Hypoxia/physiopathology , Inflammation/physiopathology , Mixed Function Oxygenases/metabolism , NF-kappa B/metabolism , Signal Transduction/physiology , Analysis of Variance , Blotting, Western , HeLa Cells , Humans , Hydroxylation , Hypoxia/metabolism , Immunoprecipitation , Inflammation/metabolism , Interleukin-1beta/metabolism , Luciferases , Mass Spectrometry , Prolyl Hydroxylases/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism
19.
J Cell Sci ; 126(Pt 6): 1454-63, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23390316

ABSTRACT

Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1α pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1α transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1α transcriptional activity, but paradoxically decreases HIF-1α stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1α to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1α transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.


Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mixed Function Oxygenases/metabolism , Models, Biological , Repressor Proteins/metabolism , Computational Biology , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/pharmacology , Oxygen/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Proteolysis , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Repressor Proteins/pharmacology , Signal Transduction , Transcriptional Activation/genetics
20.
Cell Mol Life Sci ; 71(5): 831-45, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24045706

ABSTRACT

The capacity of organisms to sense changes in the levels of internal and external gases and to respond accordingly is central to a range of physiologic and pathophysiologic processes. Carbon dioxide, a primary product of oxidative metabolism is one such gas that can be sensed by both prokaryotic and eukaryotic cells and in response to altered levels, elicit the activation of multiple adaptive pathways. The outcomes of activating CO2-sensitive pathways in various species include increased virulence of fungal and bacterial pathogens, prey-seeking behavior in insects as well as taste perception, lung function, and the control of immunity in mammals. In this review, we discuss what is known about the mechanisms underpinning CO2 sensing across a range of species and consider the implications of this for physiology, disease progression, and the possibility of developing new therapeutics for inflammatory and infectious disease.


Subject(s)
Adaptation, Biological/physiology , Carbon Dioxide/metabolism , Chemoreceptor Cells/physiology , Infections/metabolism , Inflammation/metabolism , Models, Biological , Respiration , Signal Transduction/physiology , Adenylyl Cyclases/metabolism , Animals , Aquaporins/metabolism , Carbonic Anhydrases/metabolism , Connexins/metabolism , Humans , Microbial Viability , Species Specificity
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