Daniel Crean

Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity.

The identification and dissection of cellular stress mechanisms is fundamental to understanding the susceptibility of the kidney to chemicals and pharmaceuticals and for the development of renal biomarkers indicative of sub lethal injury. Here, we utilised whole genome DNA microarrays in an attempt to uncover molecular mechanisms of response to nephrotoxin exposure. Human renal proximal tubular cells (HK-2) were treated for 12h and 48 h with 5 μM Cadmium (Cd), 30 μM Diquat Dibromide (Diq), and 5 μM Cyclosporine A (CsA). Nephrotoxin treatment resulted in an alteration of a total of 4608 transcripts. Ingenuity Pathways Analysis™ revealed the anti-oxidant and detoxification Nrf2 pathway as the most significantly enriched signaling pathway in the selected dataset. Activation of this transcription factor was confirmed as nuclear translocation and paralleled the temporal alterations of compound induced H(2)O(2) production. Transcriptomics, western blot and immunofluorescence showed an induction of both HO-1 and NQO1 with Cd and Diq exposure, but not with CsA treatment. Knockdown of Nrf2 by siRNA, reduced compound induced NQO1 mRNA to basal levels and attenuated toxin induced HO-1 mRNA expression. siRNA knock down of HO-1, but not NQO1, enhanced Cd induced H(2)O(2) production and Cd induced toxicity. Using an un-biased transcriptomic approach we have identified the Nrf2 pathway as the most significant signaling response in renal epithelial cells challenged with nephrotoxin. This study highlights the importance of this pathway and particularly HO-1 in renal epithelial adaptation to oxidative stress.

The role of HIF in immunity and inflammation.

Uncontrolled or non-resolving inflammation underpins a range of disease states including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Hypoxia is a prominent feature of chronically inflamed tissues. This is due to elevated oxygen consumption by highly metabolically active inflamed resident cells and activated infiltrating immunocytes, as well as diminished oxygen supply due to vascular dysfunction. Tissue hypoxia can have a significant impact upon inflammatory signaling pathways in immune and non-immune cells and this can impact upon disease progression. In this review, we will discuss the relationship between tissue hypoxia and inflammation and identify how hypoxia-sensitive signaling pathways are potential therapeutic targets in chronic inflammatory disease.

Development of an in vitro renal epithelial disease state model for xenobiotic toxicity testing

There is a growing impetus to develop more accurate, predictive and relevant in vitro models of renal xenobiotic exposure. As part of the EU-FP7, Predict-IV project, a major aim was to develop models that recapitulate not only normal tissue physiology but also aspects of disease conditions that exist as predisposing risk factors for xenobiotic toxicity. Hypoxia, as a common micro-environmental alteration associated with pathophysiology in renal disease, was investigated for its effect on the toxicity profile of a panel of 14 nephrotoxins, using the human proximal tubular epithelial RPTECT/TERT1 cell line. Changes in ATP, glutathione and resazurin reduction, after 14 days of daily repeat exposure, revealed a number of compounds, including adefovir dipivoxil with enhanced toxicity in hypoxia. We observed intracellular accumulation of adefovir in hypoxia and suggest decreases in the efflux transport proteins MRP4, MRP5, NHERF1 and NHERF3 as a possible explanation. MRP5 and NHERF3 were also down-regulated upon treatment with the HIF-1 activator, dimethyloxalylglycine. Interestingly, adefovir dependent gene expression shifted from alterations in cell cycle gene expression to an inflammatory response in hypoxia. The ability to investigate aspects of disease states and their influence on renal toxin handling is a key advantage of in vitro systems developed here. They also allow for detailed investigations into mechanisms of compound toxicity of potential importance for compromised tissue exposure.

M1- and M2-Type Macrophage Responses Are Predictive of Adverse Outcomes in Human Atherosclerosis.

Atherosclerosis is an inflammatory disease caused by endothelial injury, lipid deposition, and oxidative stress. This progressive disease can be converted into an acute clinical event by plaque rupture and thrombosis. In the context of atherosclerosis, the underlying cause of myocardial infarction and stroke, macrophages uniquely possess a dual functionality, regulating lipid accumulation and metabolism and sustaining the chronic inflammatory response, two of the most well-documented pathways associated with the pathogenesis of the disease. Macrophages are heterogeneous cell populations and it is hypothesized that, during the pathogenesis of atherosclerosis, macrophages in the developing plaque can switch from a pro-inflammatory (MΦ1) to an anti-inflammatory (MΦ2) phenotype and vice versa, depending on the microenvironment. The aim of this study was to identify changes in macrophage subpopulations in the progression of human atherosclerotic disease. Established atherosclerotic plaques from symptomatic and asymptomatic patients with existing coronary artery disease undergoing carotid endarterectomy were recruited to the study. Comprehensive histological and immunohistochemical analyses were performed to quantify the cellular content and macrophage subsets of atherosclerotic lesion. In parallel, expression of MΦ1 and MΦ2 macrophage markers were analyzed by real-time PCR and Western blot analysis. Gross analysis and histological staining demonstrated that symptomatic plaques presented greater hemorrhagic activity and the internal carotid was the most diseased segment, based on the predominant prevalence of fibrotic and necrotic tissue, calcifications, and hemorrhagic events. Immunohistochemical analysis showed that both MΦ1 and MΦ2 macrophages are present in human plaques. However, MΦ2 macrophages are localized to more stable locations within the lesion. Importantly, gene and protein expression analysis of MΦ1/MΦ2 markers evidenced that MΦ1 markers and Th1-associated cytokines are highly expressed in symptomatic plaques, whereas expression of the MΦ2 markers, mannose receptor (MR), and CD163 and Th2 cytokines are inversely related with disease progression. These data increase the understanding of atherosclerosis development, identifying the cellular content of lesions during disease progression, and characterizing macrophage subpopulation within human atherosclerotic plaques.

Molecular Interactions between NR4A Orphan Nuclear Receptors and NF-κB Are Required for Appropriate Inflammatory Responses and Immune Cell Homeostasis

Appropriate innate and adaptive immune responses are essential for protection and resolution against chemical, physical or biological insults. Immune cell polarization is fundamental in orchestrating distinct phases of inflammation, specifically acute phase responses followed by resolution and tissue repair. Dysregulation of immune cell and inflammatory responses is a hallmark of multiple diseases encompassing atherosclerosis, rheumatoid arthritis, psoriasis and metabolic syndromes. A master transcriptional mediator of diverse inflammatory signaling and immune cell function is NF-κB, and altered control of this key regulator can lead to an effective switch from acute to chronic inflammatory responses. Members of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors crosstalk with NF-κB to regulate immune cell function(s). Within the NR superfamily the NR4A1-3 orphan receptors have emerged as important regulators of immune cell polarization and NF-κB signaling. NR4A receptors modulate NF-κB activity in a dynamic fashion, either repressing or enhancing target gene expression leading to altered inflammatory outcome. Here we will discuss the pivotal role NR4A’s receptors play in orchestrating immune cell homeostasis through molecular crosstalk with NF-κB. Specifically, we will examine such NR4A/NF-κB interactions within the context of distinct cell phenotypes, including monocyte, macrophage, T cells, endothelial, and mesenchymal cells, which play a role in inflammation-associated disease. Finally, we review the therapeutic potential of altering NR4A/NF-κB interactions to limit hyper-inflammatory responses in vivo.

Adenosine Modulates NR4A Orphan Nuclear Receptors To Attenuate Hyperinflammatory Responses in Monocytic Cells

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.

Histamine contributes to increased RANKL to osteoprotegerin ratio through altered nuclear receptor 4A activity in human chondrocytes.

OBJECTIVE To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage. METHODS Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA. RESULTS Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression. CONCLUSION Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.

Hypoxia and inflammatory bowel disease

Inflammatory bowel disease (IBD) is a general term to describe inflammatory diseases of the gastrointestinal tract such as Crohns disease and ulcerative colitis. IBD affects approximately 1 in 200 individuals and exerts a significant health and quality of life burden on patients. Surgical intervention can be curative in ulcerative colitis but there is currently no cure for Crohns disease. Since this is the case, and the fact that patients are often diagnosed at a young age, IBD exerts a significant financial burden on the health care system, and society as a whole. The underlying pathology of IBD is complex and involves a combination of genetic, environmental and microbial factors. Regardless of the underlying causes of the condition, this disease is universally characterized by disruption to the protective epithelial barrier separating the intestinal lumen above from the mucosal immune system below. Once this barrier becomes compromised a sequence of events ensues, that can occur in repetitive cycles to ensure long-term and serious damage to the gut. The role of hypoxia and hypoxia-dependent signalling pathways are increasingly appreciated to play a role in the physiology and pathophysiology of the intestine. The intestinal epithelium normally exists in a state of physiological hypoxia, with additional tissue hypoxia a feature of active inflammatory disease. Furthermore, recent pre-clinical animal studies have clearly supported the rationale for pharmacologically manipulating the oxygen-sensitive hypoxia-inducible factor (HIF) pathway in models of IBD. Thus, this review will discuss the contribution of hypoxia sensitive pathways in the pathology of IBD. Finally we will discuss the emerging evidence for manipulation of hypoxia-sensitive pathways in the treatment of IBD.

Inhibition of protein translation as a mechanism of acidotic pH protection against ischaemic injury through inhibition of CREB mediated tRNA synthetase expression

Ischaemia associated reduction in local tissue pH is well documented but the mechanisms through which it influences cell survival remain poorly understood. Using renal epithelial HK-2 cells we demonstrate acidotic pH6.4 protects against oxygen glucose deprivation (OGD) induced cell death. Initial exploration of the mechanisms responsible using microarray analysis revealed acidotic inhibition of OGD induced aminoacyl-tRNA synthetase (ARS) gene expression. These genes are key components of protein translation, which was markedly attenuated by reduced pH. Inhibition of protein synthesis using the ARS inhibitor halofuginone or cycloheximide protected against OGD induced injury. To explore further we focussed on the transcription factor CREB, identified by pathway analysis of microarray data and observed a pH dependent decrease in OGD induced activation. Inhibition of CREB/CBP interaction prevented OGD induced isoleucyl-ARS (IARS) expression, reduced protein synthesis and protected against OGD induced cellular injury. In addition we also observed that acidotic pH attenuated the OGD induced pro-apoptotic unfolded protein response (UPR) activated gene DDIT3. We suggest that maladaptive activation of CREB and ARS gene expression, through the maintenance of protein synthesis contributes to ER stress and UPR activation and that acidotic pH through inhibition of CREB activation inhibits protein synthesis and ultimately UPR activated apoptotic signals.

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1–3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1–3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.