Corticotropin-releasing factor regulates TLR4 expression in the colon and protects mice from colitis.

BACKGROUND & AIMS: Defects in the colonic innate immune response have been associated with inflammatory bowel disease (IBD). Corticotropin-releasing hormone (CRH, or corticotropin-releasing factor [CRF]) is a neuropeptide that mediates the stress response in humans, is an immunomodulatory factor with proinflammatory effects, and regulates transcription of Toll-like receptors (TLR)-2 and TLR4. We investigated the role of CRF in an innate immunity-dependent mouse model of IBD. METHODS: Crh(-/-) and wild-type (Crh(+/+)) mice, which are glucocorticoid insufficient, were given dextran sodium sulfate in their drinking water to induce colitis; in some experiments, mice were also given glucocorticoids. Phenotypes of mice were compared; tissues were analyzed by histology and for expression of immune mediators. RESULTS: Crh(-/-) mice had more colonic inflammation than Crh(+/+) mice, characterized by reduced numbers of crypts and severe epithelial damage and ulcerations. Colonic tissue levels of the proinflammatory factors interleukin-12 and prostaglandin E(2) were increased in the Crh(-/-) mice. Colons of Crh(-/-) mice expressed lower levels of Tlr4 than wild-type mice before, but not after, colitis was induced. Administration of glucocorticoid at low levels did not prevent Crh(-/-) mice from developing severe colitis. Crh(-/-) mice were unable to recover from acute colitis, as indicated by their increased death rate. CONCLUSIONS: Mice deficient in CRF down-regulate TLR4 and are more susceptible to dextran sodium sulfate-induced colitis. CRF has anti-inflammatory effects in innate immunity-dependent colitis and its recovery phase; these are independent of glucocorticoid administration. CRF might therefore be developed as a therapeutic target for patients with IBD.

Structural domains determining signalling characteristics of the CRH-receptor type 1 variant R1beta and response to PKC phosphorylation.

Mammalian adaptive mechanisms to stressful stimuli involve release of corticotropin-releasing hormone (CRH) and downstream activation of specific G-protein-coupled 7 transmembrane domain receptors. These CRH receptors (CRH-R) are expressed as multiple mRNA spliced variants. In contrast to other mammals, the human type 1 CRH-R gene contains an additional exon (exon 6) that needs to be spliced out in order to generate the fully active CRH-R1alpha. Transcription of all 14 exons results in a CRH-R1 variant (CRH-R1beta) with an extended 1st intracellular loop (IC1); this sequence modification impairs signalling activity and alters receptor responsiveness to PKC-induced phosphorylation that leads to signalling desensitization and receptor endocytosis. To elucidate structure-function relationships and delineate sequences involved in CRH-R1beta properties, site directed mutagenesis was used to introduce a number of specific mutations into IC1 of CRH-R1beta as well as replace specific phospho-acceptor residues within the aminoacid sequence of CRH-R1alpha and CRH-R1beta. Mutant receptors were transiently expressed in human embryonic kidney (HEK293) cells and tested for their abilities to increase intracellular cAMP and their response to PKC-induced phosphorylation. Results identified a penta-aminoacid cassette within the 29-aminoacid insert of CRH-R1beta, which contains multiple positive charged aminoacids (F170-R174), as an important structural determinant for the impaired cAMP response. Furthermore, serine at position 408 in the carboxy-terminus appears to be important for mediating CRH-R1alpha resistance, but not CRH-R1beta susceptibility, to PKC-induced desensitization and internalization. These findings provide new insights about the structural determinants of CRH-R1 coupling to Gs proteins and response to protein kinase phosphorylation.

Regulation of appetite and insulin signaling in inflammatory states.

Inflammatory states are characterized by decreased food intake, hyperglycemia, and insulin resistance. The contribution of cytokines in this phenotype is important and is exerted through activation of SOCS proteins and inhibition of insulin signaling, as well as through direct stimulation of the ob gene. Obesity, a condition that has reached epidemic rates, is characterized by hyperglycemia, hyperlipidemia, insulin resistance and increased food intake, and body weight. In the following article we summarize the current views of the mechanisms underlying insulin resistance in obesity and the other inflammatory states. We also discuss the regulation of appetite in inflammatory states, and we provide evidence on the cytokine-independent induction of anorexia following immune activation in mice. Understanding of the exact mechanisms regulating these processes may provide important insights for the control of this group of diseases that compromise to a great extent the quality of life and are associated with high mortality.

Regulation of corticotropin-releasing hormone receptor type 1alpha signaling: structural determinants for G protein-coupled receptor kinase-mediated phosphorylation and agonist-mediated desensitization.

Attenuation of CRH receptor type 1 (CRH-R1) signaling activity might involve desensitization and uncoupling of CRH-R1 from intracellular effectors. We investigated the desensitization of native CRH-R in human myometrial cells from pregnant women and recombinant CRH-R1alpha stably overexpressed in human embryonic kidney (HEK) 293 cells. In both cell types, CRH-R1-mediated adenylyl cyclase activation was susceptible to homologous desensitization induced by pretreatment with high concentrations of CRH. Time course studies showed half-maximal desensitization occurring after approximately 40 min of pretreatment and full recovery of CRH-R1alpha functional response within 2 h of removal of CRH pretreatment. In HEK 293 cells, desensitization of CRH-R1alpha was associated with receptor phosphorylation and subsequent endocytosis. To analyze the mechanism leading to CRH-R1alpha desensitization, we overexpressed a truncated beta-arrestin (319-418) and performed coimmunoprecipitation and G protein-coupled receptor kinase (GRK) translocation studies. We found that GRK3 and GRK6 are the main isoforms that interact with CRH-R1alpha, and that recruitment of GRK3 requires Gbetagamma-subunits as well as beta-arrestin. Site-directed mutagenesis of Ser and Thr residues in the CRH-R1alpha C terminus, identified Thr399 as important for GRK-induced receptor phosphorylation and desensitization.We conclude that homologous desensitization of CRH-R1alpha involves the coordinated action of multiple GRK isoforms, Gbeta gamma dimers and beta-arrestin. Based on our identification of key amino acid(s) for GRK-dependent phosphorylation, we demonstrate the importance of the CRH-R1alpha carboxyl tail for regulation of receptor activity.

Mechanisms of obesity and related pathology: linking immune responses to metabolic stress.

There is a tightly regulated interaction, which is well-conserved in evolution, between the metabolic and immune systems that is deranged in states of over- or under-nutrition. Obesity, an energy-rich condition, is characterized by the activation of an inflammatory process in metabolically active sites such as adipose tissue, liver and immune cells. The consequence of this response is a sharp increase in circulating levels of proinflammatory cytokines, adipokines and other inflammatory markers. Activation of the immune response in obesity is mediated by specific signaling pathways, with Jun N-terminal kinase and IkappaB kinase beta/nuclear factor kappa-light-chain-enhancer of activated B cells being the most well studied. It is known that the above events modify insulin signaling and result in the development of insulin resistance. The nutrient overload characterizing obesity is a metabolic stressor associated with intracellular organelle (e.g. the endoplasmic reticulum) stress. The exact characterization of the series of events and the mechanisms that integrate the inflammatory response with metabolic homeostasis at the cellular and systemic level is a very active research field. In this minireview, we discuss the signaling pathways and molecules associated with the development of obesity-induced inflammation, as well as the evidence that supports a critical role for the stress response in this process.

Differential responses of corticotropin-releasing hormone receptor type 1 variants to protein kinase C phosphorylation.

Corticotropin-releasing hormone (CRH) regulates diverse biological functions in mammals, through activation of two types of specific G protein-coupled receptors that are expressed as multiple mRNA spliced variants. In most cells, the type 1alpha CRH receptor (CRH-R1alpha) preferentially activates the G(s)-adenylyl cyclase signaling cascade. CRH-R1alpha-mediated signaling activity is impaired by insertion of 29 amino acids in the first intracellular loop, a sequence modification that is characteristic of the human-specific CRH-R1beta variant. In various tissues, CRH signaling events are regulated by protein kinase C (PKC). The CRH receptors contain multiple putative PKC phosphorylation sites that represent potential targets. To investigate this, we expressed recombinant CRH-R1alpha or CRH-R1beta in human embryonic kidney 293 cells and analyzed signaling events after PKC activation. Agonist (oxytocin) or phorbol 12-myristate 13-acetate-induced activation of PKC led to phosphorylation of both CRH-R1 variants. However, CRH-R1alpha and CRH-R1beta exhibited different functional responses to PKC-induced phosphorylation, with only the CRH-R1beta susceptible to cAMP signaling desensitization. This was associated with a significant decrease of accessible CRH-R1beta receptors expressed on the cell surface. Both CRH-R1 variants were susceptible to homologous desensitization and internalization following treatment with CRH; however, PKC activation increased internalization of CRH-R1beta but not CRH-R1alpha in a beta-arrestin-independent manner. Our findings indicate that CRH-R1alpha and -R1beta exhibit differential responses to PKC-induced phosphorylation, and this might represent an important mechanism for functional regulation of CRH signaling in target cells.

EphB1-mediated cell migration requires the phosphorylation of paxillin at Tyr-31/Tyr-118.

Interactions between Eph receptors and their membrane-bound ligands (ephrins) are of critical importance for key developmental processes such as boundary formation or vascular development. Their downstream signaling pathways are intricate and heterogeneous at several levels, the combined effect being a highly complex and flexible system. Here we demonstrate that activated EphB1 induces tyrosine phosphorylation of the focal adhesion protein paxillin at Tyr-31 and Tyr-118 and is recruited to paxillin-focal adhesion kinase (FAK) complexes. Pretreatment with the specific Src inhibitor PP2, or expression of dominant-negative, kinase-dead c-Src abrogates EphB1-induced tyrosine phosphorylation of paxillin. Cells transfected with the paxillin mutant Y31F/Y118F displayed a reduced migration in response to ephrin B2 stimulation. Furthermore, expression of an LD4 deletion mutant (paxillin DeltaLD4) significantly reduces EphB1-paxillin association, paxillin tyrosine phosphorylation, as well as EphB1-dependent cell migration. Finally, mutation of the Nck-binding site of EphB1 (Y594F) interrupts the interaction between Nck, paxillin, and EphB1. These data suggest a model in which ligand-activated EphB1 forms a signaling complex with Nck, paxillin, and focal adhesion kinase and induces tyrosine phosphorylation of paxillin in a c-Src-dependent manner to promote cell migration.