Marko Radulovic

Deletion of Crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2

Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G-protein–coupled receptors, Crhr1 (refs 3–5) and Crhr2 (refs 6–9), causing (among other transductional events) phosphorylation of the transcription factor Creb (ref. 10). The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1 (refs 11,12). The enhanced anxiety of Crhr2-deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2−/− mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2-deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender.

Pharmacology and biology of corticotropin-releasing factor (CRF) receptors.

The biology of corticotropin-releasing factor (CRF) finds increasing interest in the scientific community because of the neuromodulatory actions of CRF on brain functions such as learning, anxiety, feeding, and locomotion. Additional actions on immunumodulation and apoptosis have recently been discovered. All actions of CRF are mediated by G protein-coupled receptors, which trigger different, sometimes opposite actions in different regions of the central nervous system. The CRF system exhibits considerable plasticity by the involvement of numerous different ligands, splice variants, and transductional couplings. The generation of multiple splice variants is facilitated by the intron exon structure of the CRF receptor genes.

Differential activation of CRF receptor subtypes removes stress-induced memory deficit and anxiety.

The objective of this study was to investigate the role of corticotropin‐releasing factor receptors 1 (CRF1) and 2 (CRF2) in anxiety‐like behavior and learning of C57BL/6J mice after exposure to a stressful stimulus. When C57BL/6J mice were exposed to immobilization (1 h) serving as stressful stimulus, context‐ and tone‐dependent fear conditioning were impaired if the training followed immediately after immobilization. The stress‐induced impairment of context‐dependent fear conditioning was prevented by specific blockade of CRF2 of the lateral septum (LS) with anti‐sauvagine‐30. Immobilization did not only affect conditioned fear, but also enhanced, through CRF2 of the LS, anxiety‐like behavior determined with the elevated plus maze. Recovery from stress‐induced anxiety and impairment of context‐dependent fear conditioning was observed after 1 h delay of training and required hippocampal CRF1, as indicated by the finding that this recovery was prevented by blockade of intrahippocampal CRF1. It was concluded that exposure to a stressor initially affected both anxiety‐like behavior and contextual conditioned fear through septal CRF2, while the later activation of hippocampal CRF1 resulted in the return to baseline levels of both processes. Intraventricular injection of mouse urocortin 2, a CRF2‐selective agonist, removed the stress‐induced anxiety and learning impairment, but did not reduce the activation of the hypothalamic pituitary adrenal axis indicative of the hormonal stress response. We propose that the enhanced anxiety is the component of the stress response responsible for the memory deficit.

Corticotropin-releasing factor (CRF) rapidly suppresses apoptosis by acting upstream of the activation of caspases.

The physiological role of the corticotropin‐releasing factor (CRF) family of peptides has recently been extended by emerging evidence of their cytoprotective effects. To determine whether CRF‐mediated cytoprotection is linked to caspase‐dependent apoptosis, the effect of CRF on the activation of caspases was investigated in detail in Y79 human retinoblastoma cells. The results presented here demonstrate that the cytoprotective effect of CRF against the actions of camptothecin (CT) was mediated by CRF receptor subtype 1, but not subtype 2. The observed CRF‐mediated cytoprotection involved rapid and pronounced suppression of proteolytic processing and activation of procaspase‐3, exerted even when CRF was added hours after the application of the cytotoxic agent. Surprisingly, activation of procaspase‐3 preceded activation of the initiator procaspases 2, 8, 9 and 10 during CT‐induced apoptosis of Y79 cells. The mechanism of the effect of CRF was examined using inhibitors of signalling pathways such as Wortmannin (Akt), cyclic AMP‐dependent protein kinase (PKA), extracellular signal‐regulated kinase (ERK), protein kinase c (PKC), p38 mitogen‐activated protein kinase (p38 MAPK), phospholipase c (PLC), nuclear factor‐κB (NF‐κΒ) and c‐jun N‐terminal kinase (JNK). The involvement of PKA in the mediation of the anti‐apoptotic effect of CRF has been established. Taken together, these results demonstrate for the first time that the cytoprotective effect of CRF involved suppression of pro‐apoptotic pathways at a site upstream of activation of procaspase‐3.

CRF and CRF Receptors

Corticotropin-releasing factor (CRF), a 41-residue polypeptide, is one of the hypothalamic hypophysiotropic peptides. In mammals, CRF is synthesized in the hypothalamic paraventricular nucleus PVN, transported to the median eminence and secreted in the portal circulation reaching the corticotrophs of the anterior pituitary (Cummings et al. 1983; Swanson et al. 1983), where it stimulates the release of corticotropin which then enhances the release of cortisol from the adrenal cortex. Hypothalamus, pituitary and adrenal are thus functionally connected and constitute the hypothalamus pituitary adrenal (HPA) axis. Activation of this axis represents an important indicator of the response to stress. On this basis, the investigation of the CRF biology has been important for both basic scientists and clinicians. It has been speculated that CRF may play a role in depressive illness (Mitchell 1998) and cognitive disease (reviewed by De Souza 1995). The characterization of the primary structure of CRF (Spiess et al. 1981) purified on the basis of its hypophysiotropic activity (Vale et al. 1981) enabled rapid progress in the understanding of the physiology, biochemistry and anatomy of CRF in the brain of numerous animal species.

Spatial distribution of cellular function: the partitioning of proteins between mitochondria and the nucleus in MCF7 breast cancer cells.

Concurrent proteomics analysis of the nuclei and mitochondria of MCF7 breast cancer cells identified 985 proteins (40% of all detected proteins) present in both organelles. Numerous proteins from all five complexes involved in oxidative phosphorylation (e.g., NDUFA5, NDUFB10, NDUFS1, NDUF2, SDHA, UQRB, UQRC2, UQCRH, COX5A, COX5B, MT-CO2, ATP5A1, ATP5B, ATP5H, etc.), from the TCA-cycle (DLST, IDH2, IDH3A, OGDH, SUCLAG2, etc.), and from glycolysis (ALDOA, ENO1, FBP1, GPI, PGK1, TALDO1, etc.) were distributed to both the nucleus and mitochondria. In contrast, proteins involved in nuclear/mitochondrial RNA processing/translation and Ras/Rab signaling showed different partitioning patterns. The identity of the OxPhos, TCA-cycle, and glycolysis proteins distributed to both the nucleus and mitochondria provides evidence for spatio-functional integration of these processes over the two different subcellular organelles. We suggest that there are unrecognized aspects of functional coordination between the nucleus and mitochondria, that integration of core functional processes via wide subcellular distribution of constituent proteins is a common characteristic of cells, and that subcellular spatial integration of function may be a vital aspect of cancer.

The effect of acute immobilization stress on the abundance of corticotropin-releasing factor receptor in lymphoid organs

We have previously found a dramatic increase of corticotropin-releasing factor receptor (CRF-R1) production in splenic neutrophils of male C57BL/6N mice after application of an immunological stimulus. We demonstrate here that immobilization, a predominantly psychological stress, exhibited a similar effect. Shortly after 90 min of immobilization, the number of splenic CRF-RI+ cells was transiently increased by nearly 8-fold, while it was reduced in thymus and unchanged in lymph nodes. The CRF-R1+ cells were detected by an affinity-purified polyclonal antibody directed against the N-terminus of CRF-R1, and identified as neutrophils, eosinophils or their immature precursors on the basis of their nuclear shapes, Wright-Giemsa staining and colocalization of CRF-R1 with the ER-MP58 antigen.

CKS Proteins Protect Mitochondrial Genome Integrity by Interacting with Mitochondrial Single-stranded DNA-binding Protein

Cyclin-dependent kinase subunit (CKS) proteins interact with cyclin-dependent kinases (CDKs) with high affinity. Mammalian CKS1 and CKS2 bind CDK1 and CDK2 and partake in the control of cell cycle progression. We identified CKS-interacting proteins by affinity purification followed by mass spectrometry in the human lymphocytic cell line Ramos. Apart from known interactors, such as CDKs, we identified a novel CDK-dependent interaction between CKS proteins and the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB bound both CKS1 and CKS2 and underwent CDK-dependent phosphorylation. mtSSB is known to participate in replication of mitochondrial DNA. We demonstrated that mitochondrial morphology and DNA integrity were compromised in cells depleted of both CKS proteins or that had inhibited CDK activity. These features are consistent with the hypothesis of CKS-dependent regulation of mtSSB function and support a direct role of cell cycle proteins in controlling mitochondrial DNA replication.

Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

We have used a subcellular spatial razor approach based on LC-MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function.

Systematic Nucleo-Cytoplasmic Trafficking of Proteins Following Exposure of MCF7 Breast Cancer Cells to Estradiol

We have used a proteomics subcellular spatial razor approach to look at changes in total protein abundance and in protein distribution between the nucleus and cytoplasm following exposure of MCF7 breast cancer cells to estradiol. The dominant response of MCF7 cells to estrogen stimulation involves dynamic changes in protein subcellular spatial distribution rather than changes in total protein abundance. Of the 3604 quantitatively monitored proteins, only about 2% show substantial changes in total abundance (>2-fold), whereas about 20% of the proteins show substantial changes in local abundance and/or redistribution of their subcellular location, with up to 16-fold changes in their local concentration in the nucleus or the cytoplasm. We propose that dynamic redistribution of the subcellular location of multiple proteins in response to stimuli is a fundamental characteristic of cells and suggest that perturbation of cellular spatial control may be an important feature of cancer.