The unliganded aryl hydrocarbon receptor (AHR) is found in a complex with other proteins including the 90-kDa heat shock protein (Hsp90) and a 37-kDa protein we refer to as ARA9. We found that the three tetratricopeptide repeats found in the COOH terminus of ARA9 are necessary and sufficient for interaction with the AHR complex. Conversely, the AHR's "repressor"/Hsp90 binding domain is required for interaction with ARA9. Because ARA9 closely resembles the 52-kDa FK506-binding protein (FKBP52), found in the unliganded glucocorticoid receptor (GR) complex, we compared the binding specificities of ARA9 and FKBP52 for AHR and GR. In co-immunoprecipitation experiments, ARA9 specifically associated with AHR-Hsp90 complex but not with GRHsp90 complexes. In addition, ARA9 showed a greater capacity than FKBP52 to associate with AHR-Hsp90 complexes. The biological importance of this interaction was suggested by the observation that in a yeast expression system ARA9 expression enhanced the response of AHR to the agonist -napthoflavone, decreasing the EC 50 by greater than 5-fold and increasing the maximal response 2.5-fold. In contrast, co-expression of FKBP52 had no effect on AHR signaling. In addition, although ARA9 contains a domain similar to that found in other FK506-binding proteins, ARA9 binding to 3 H-FK506 could not be detected. Finally, we have characterized the developmental and expression pattern of ARA9 in the developing mouse embryo and mapped the ARA9 locus to human chromosome 11q13.3.
Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1alpha, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1alpha -/- cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1alpha -/- cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1alpha is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses.
G-protein coupled receptor kinase-5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G-protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non-GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll-like receptor-4-induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5 −/− ) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)-induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS-induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5 −/− mice compared to the GRK5 +/+ mice. Similar to the in vivo studies, primary macrophages from GRK5 −/− mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4-induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS-induced IκBα phosphorylation, NFκB p65 nuclear translocation and NFκB binding were markedly attenuated in GRK5 −/− macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4-induced IκBα-NFκB pathway as well as a key modulator of lipopolysaccharide-induced inflammatory response.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of agonists like 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the current model for AHR signaling, the unliganded receptor is found in the cytosol as part of a complex with a dimer of the 90-kDa heat shock protein and an immunophilin-like molecule, ARA9. In yeast, expression of ARA9 results in an increase in the maximal agonist response and a leftward shift in the AHR dose-response curve. To better understand the mechanism by which ARA9 modifies AHR signal transduction, we performed a series of coexpression experiments in yeast and mammalian cells. Our results demonstrate that ARA9's influence on AHR signaling is not due to inhibition of a membrane pump or modification of the receptor's transactivation properties. Using receptor photoaffinity labeling experiments, we were able to show that ARA9 enhances AHR signal transduction by increasing the available AHR binding sites within the cytosolic compartment of the cell. Our evidence suggests that ARA9's effects are related to its role as a cellular chaperone; i.e. we observed that expression of ARA9 increases the fraction of AHR in the cytosol and also stabilized the receptor under heat stress. The AHR1 is a ligand-activated transcription factor that mediates the biological effects of halogenated dioxins and related compounds (1). In a widely held model of dioxin signal transduction, the AHR is found in the cytosol, in a complex with Hsp90 and a newly discovered protein known as ARA9 (2-4).2 In the presence of agonist, the AHR translocates to the nucleus, where it binds to its nuclear partner, ARNT. This AHR⅐ARNT heterodimer is capable of binding DNA and promotes the transcription of a battery of responsive genes including those encoding a number of xenobiotic-metabolizing enzymes (5). Hsp90 has been shown to play a role in maintaining AHR in a conformation that can bind ligand with high affinity (6 -8). Although ARA9 has been shown to increase AHR function in yeast and mammalian cells, its role in AHR signaling is not understood (7-10).ARA9 was initially identified in yeast two-hybrid screens, in which the AHR or the hepatitis B virus protein X were used as bait (9 -11). Later, it was purified from monkey cells in a complex with the AHR (12). ARA9 contains two notable structural motifs. In its amino terminus, ARA9 contains an FKBP homology domain. This domain shares 28% amino acid sequence identity to FKBP12 (9,11,12). However, ARA9 is unable to bind FK506 and does not appear to have peptidyl prolyl isomerase activity 3 (13). In its carboxyl terminus, ARA9 contains three TPRs. TPRs are defined by strings of 34 amino acids that have been shown to play roles in protein-protein interaction (14). This domain structure is similar to that found in the GR-associated immunophilin, FKBP52, which contains two FKBP domains in its amino terminus and three TPRs in its carboxyl terminus (15,16). In addition to their structural similarities, ARA9 and FKBP52 are found associated to...
Neonatal respiratory distress syndrome (RDS) is mainly the result of perturbation in surfactant production and is a common complication seen in premature infants. Normal fetal lung development and alveolar cell differentiation is regulated by a network of transcription factors. Functional loss of any of these factors will alter the developmental program and impact surfactant production and normal gas exchange. During development, the fetus is exposed to varying oxygen concentrations and must be able to quickly adapt to these changes in order to survive. Hypoxia-inducible factor 1␣ (HIF1␣) is the primary transcription factor that is responsible for regulating the cellular response to changes in oxygen tension and is essential for normal development. Its role in lung maturation is not well defined and to address this knowledge gap, a lung-specific HIF1␣ knock-out model has been developed. Loss of HIF1␣ early in lung development leads to pups that die within hours of parturition, exhibiting symptoms similar to RDS. Lungs from these pups display impaired alveolar epithelial differentiation and an almost complete loss of surfactant protein expression. Ultrastructural analysis of lungs from HIF1␣ deletion pups had high levels of glycogen, aberrant septal development, and decreased expression of several factors necessary for proper lung development, including HIF2␣, -catenin, and vascular endothelial growth factor. These results suggest that HIF1␣ is essential for proper lung maturation and alteration in its normal signaling during premature delivery might explain the pathophysiology of neonatal RDS.During development, an embryo is exposed to varying levels of oxygen as a balance is created between vascularization and tissue growth. Localized hypoxia, a decrease in available oxygen, is a normal part of this process. The programmed responses to these decreases in available oxygen are essential for viability (1, 2). In utero, the embryo is supplied with oxygen and nutrients through the placental barrier. Following parturition, oxygen is supplied by the neonatal lungs and therefore, proper intrauterine development of the alveolar gas exchange regions of the lung is essential for the newborn's first breath and for sustaining life outside the womb (3).Lung morphogenesis is a complex process that is orchestrated by several transcription factors, growth factors, and extracellular cues (4). For example, thyroid transcription factor-1 regulates the expression of the genes for Clara cell secretory protein (CCSP) 2 produced in Clara cells in the tracheobronchial airways and various surfactants produced by alveolar type II cells in the lung parenchyma (5, 6). CCAAT-enhancerbinding protein ␣ (CEBP␣) is essential for proper regulation of alveolar Type II cell differentiation, and Forkhead box A2 (Foxa2) controls various cellular programs involved in lung development (e.g. surfactant expression) (6 -8). Ablation of any of these factors results in major structural and functional abnormalities ranging from undeveloped alveolar structu...
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