Intense impact of IL-1ß expressing inflammatory macrophages in acute aortic dissection.

There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and ß-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1ß (IL-1ß) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1ß antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1ß in both human AAD samples and in a murine AAD model. Anti-IL-1ß antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.

Chronic stress alters lipid mediator profiles associated with immune-related gene expressions and cell compositions in mouse bone marrow and spleen.

Despite the importance of lipid mediators in stress and depression and their link to inflammation, the influence of stress on these mediators and their role in inflammation is not fully understood. This study used RNA-seq, LC-MS/MS, and flow cytometry analyses in a mouse model subjected to chronic social defeat stress to explore the effects of acute and chronic stress on lipid mediators, gene expression, and cell population in the bone marrow and spleen. In the bone marrow, chronic stress induced a sustained transition from lymphoid to myeloid cells, accompanied by corresponding changes in gene expression. This change was associated with decreased levels of 15-deoxy-d12,14-prostaglandin J2, a lipid mediator that inhibits inflammation. In the spleen, chronic stress also induced a lymphoid-to-myeloid transition, albeit transiently, alongside gene expression changes indicative of extramedullary hematopoiesis. These changes were linked to lower levels of 12-HEPE and resolvins, both critical for inhibiting and resolving inflammation. Our findings highlight the significant role of anti-inflammatory and pro-resolving lipid mediators in the immune responses induced by chronic stress in the bone marrow and spleen. This study paves the way for understanding how these lipid mediators contribute to the immune mechanisms of stress and depression.

Overexpression of NT-3 in the hippocampus suppresses the early phase of the adult neurogenic process.

The dentate gyrus (DG) of the hippocampus regulates stress-related emotional behaviors and ensures neurogenesis throughout life. Neurotrophin-3 (NT-3) is a neurotrophic factor that regulates neuronal differentiation, survival, and synaptic formation in both the peripheral and central nervous systems. NT-3 is expressed in the adult DG of the hippocampus; several chronic stress conditions enhance NT-3 expression in rodents. However, functional modulation of the adult DG by NT-3 signaling remains unclear. To directly investigate the impact of NT-3 on DG function, NT-3 was overexpressed in the hippocampal ventral DG by an adeno-associated virus carrying NT-3 (AAV-NT-3). Four weeks following the AAV-NT-3 injection, high NT-3 expression was observed in the ventral DG. We examined the influence of NT-3 overexpression on the neuronal responses and neurogenic processes in the ventral DG. NT-3 overexpression significantly increased the expression of the mature DG neuronal marker calbindin and immediate early genes, such as Fos and Fosb, thereby suggesting DG neuronal activation. During neurogenesis, the number of proliferating cells and immature neurons in the subgranular zone of the DG significantly decreased in the AAV-NT-3 group. Among the neurogenesis-related factors, Vegfd, Lgr6, Bmp7, and Drd1 expression significantly decreased. These results demonstrated that high NT-3 levels in the hippocampus regulate the activation of mature DG neurons and suppress the early phase of neurogenic processes, suggesting a possible role of NT-3 in the regulation of adult hippocampal function under stress conditions.

N-Acetylcysteine Suppresses Microglial Inflammation and Induces Mortality Dose-Dependently via Tumor Necrosis Factor-α Signaling.

N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.

Mobilization efficiency is critically regulated by fat via marrow PPARδ.

The mobilization efficiency of hematopoietic stem/progenitor cells from bone marrow (BM) to circulation by granulocyte colony-stimulating factor (G-CSF) is dramatically dispersed in humans and mice with no mechanistic lead for poor mobilizers. The regulatory mechanism for mobilization efficiency by dietary fat was assessed in mice. Fat-free diet (FFD) for 2 weeks greatly increased mobilization compared to normal diet (ND). The BM mRNA level of peroxisome proliferator-activated receptor δ (PPARδ), a receptor for lipid mediators, was markedly up-regulated by G-CSF in mice fed with ND and displayed strong positive correlation with widely scattered mobilization efficiency. It was hypothesized that BM fat ligand for PPARδ might inhibit mobilization. The PPARδ agonist inhibited mobilization in mice fed with ND and enhanced mobilization by FFD. Treatment with the PPARδ antagonist and chimeric mice with PPARδ+/- BM showed enhanced mobilization. Immunohistochemical staining and flow cytometry revealed that BM PPARδ expression was enhanced by G-CSF mainly in mature/immature neutrophils. BM lipid mediator analysis revealed that G-CSF treatment and FFD resulted in the exhaustion of ω3-polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). EPA induced the up-regulation of genes downstream of PPARδ, such as carnitine palmitoyltransferase-1α and angiopoietin-like protein 4 (Angptl4), in mature/immature neutrophils in vitro and inhibited enhanced mobilization in mice fed with FFD in vivo. Treatment of wild-type mice with the anti-Angptl4 antibody enhanced mobilization together with BM vascular permeability. Collectively, PPARδ signaling in BM mature/immature neutrophils induced by dietary fatty acids negatively regulates mobilization, at least partially, via Angptl4 production.

Stress-induced sleep-like inactivity modulates stress susceptibility in mice.

Severe environmental and social stress induces dysregulation of sleep along with mood and cognitive disturbances. However, the role and mechanism of this sleep dysregulation remain elusive. Here we evaluated sleep-like inactivity measured by voluntary movements and its relationship to social behaviors in mice without or with social defeat stress as well as the stressed mice with subsequent sleep deprivation. Social defeat stress immediately induced sleep-like inactivity with decreased body temperature. In the social interaction test, the control mice showed high social interest and its correlation with social sniffing intensity, the latter of which indicates positive valence of social sniffing. After the stress, these social characteristics were maintained in stress-resilient mice, but disrupted in stress-susceptible mice, leading to social avoidance. Sleep deprivation after the stress decreased social sniffing intensity along with reduced social interest, but enhanced the exploratory activity with the positive valence of social sniffing. We also found by c-Fos immunohistochemistry that the stress activated sleep-related brain regions, the dorsomedial hypothalamus and ventrolateral periaqueductal gray. Collectively, these findings show that stress activates sleep-related brain regions and induces sleep-like inactivity, contributing to multiple roles of stress-induced sleep for social behaviors.

Social defeat stress-specific increase in c-Fos expression in the extended amygdala in mice: Involvement of dopamine D1 receptor in the medial prefrontal cortex.

We recently reported that dopamine D1 receptor in the medial prefrontal cortex (mPFC) is activated by subthreshold social defeat stress and suppresses the induction of depressive-like behavior in mice. However, which mPFC projection(s) mediates this antidepressant-like effect remains poorly understood. Here we show that social defeat stress specifically increased c-Fos expression, a marker for neuronal activity, in distinct brain regions involved in emotional regulation, relative to novelty-induced exploration. Among these brain areas, D1 knockdown in the mPFC decreased social defeat stress-induced c-Fos expression in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a subregion of the extended amygdala. Using retrograde adeno-associated virus vectors and transgenic mice expressing Cre recombinase under the D1 promoter, we also found that D1-expressing deep-layer pyramidal neurons in the mPFC send direct projections to the IPAC. These findings indicate that social defeat stress specifically activates neurons in distinct brain areas, among which the IPAC is regulated by dopamine D1 receptor in the mPFC perhaps through direct projections. Thus, this study provides hints toward identifying neural circuits that underlie antidepressant-like effects of stress-induced dopamine D1 receptor signaling in the mPFC.

Roles of multiple lipid mediators in stress and depression.

Prolonged or excessive stress may induce emotional and cognitive disturbances, and is a risk factor for mental illnesses. Using rodent chronic stress models of depression, roles of multiple lipid mediators related to inflammation have been revealed in chronic stress-induced emotional alterations. Prostaglandin (PG) E2, an arachidonic acid (AA)-derived lipid mediator, and its receptor subtype EP1 mediate depression-like behavior induced by repeated social defeat stress through attenuating prefrontal dopaminergic activity. Repeated social defeat stress activates microglia through innate immune receptors, and induces PGE2 synthesis through cyclooxygenase-1, a prostaglandin synthase enriched in microglia. PGD2, another AA-derived lipid mediator, has been implicated in depression induced by chronic stress, although either pro-depressive or anti-depressive actions have been reported. Chronic stress up-regulates hippocampal expression of 5-lipoxygenase, hence synthesis of cysteinyl leukotrienes, thereby inducing depression through their receptors. Consistent with beneficial effects of n-3 fatty acids in the diet of depressive patients, resolvins-a novel class of pro-resolving lipid mediators-in the brain attenuate neuroinflammation-associated depression. These findings in animal models of depression offer lipid mediators and related molecules as novel therapeutic targets for treating depression. To translate these findings into clinics, translational biomarkers to visualize lipid mediator profiles in depressive patients need to be established.

Ulk2 controls cortical excitatory-inhibitory balance via autophagic regulation of p62 and GABAA receptor trafficking in pyramidal neurons.

Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.

G-CSF-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via PGE2.

Granulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by ß-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all ß-adrenergic receptors, only ß3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced ß-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.

Characterization of Tamoxifen as an Antifungal Agent Using the Yeast Schizosaccharomyces Pombe Model Organism.

Tamoxifen, a selective estrogen receptor modulator used for managing breast cancer, is known to have antifungal activity. However, its molecular mechanism remains unknown. Using the fission yeast Schizosaccharomyces pombe as a model organism, we have explored the mechanism involved in antifungal action of tamoxifen. Since tamoxifen was shown to inhibit the binding of calmodulin to calcineurin in fungi, we first examined involvement of these molecules and found that overexpression of a catalytic subunit of calcineurin and its constitutively active mutant as well as calmodulin increases tamoxifen sensitivity. Since terbinafine and azoles inhibit enzymes for ergosterol biosynthesis, Erg1 and Erg11, for their antifungal actions, we also examined involvement of these molecules. Overexpression of Erg1 and Erg11 reduced the sensitivity to terbinafine and azoles, respectively, but increased tamoxifen sensitivity, suggesting that ergosterol biosynthesis is differently related to the action of tamoxifen and those of terbinafine and azoles. To elucidate molecules involved in tamoxifen action, we performed a genome-wide screen for altered sensitivity to tamoxifen using a fission yeast gene deletion library, and identified various hypersensitive and resistant mutants to this drug. Notably, these mutants are rarely overlapped with those identified in similar genetic screens with currently used antifungals, suggesting a novel mode of antifungal action. Furthermore, tamoxifen augmented antifungal actions of terbinafine and azoles, suggesting synergetic actions between these drugs. Therefore, our findings suggest that calmodulin-calcineurin pathway and ergosterol biosynthesis are related to antifungal action of tamoxifen, and propose novel targets for antifungal development as well as combined therapy with tamoxifen for fungal diseases.

Prostaglandin E receptor EP1 forms a complex with dopamine D1 receptor and directs D1-induced cAMP production to adenylyl cyclase 7 through mobilizing G(ßγ) subunits in human embryonic kidney 293T cells.

The mechanism underlying the crosstalk between multiple G protein-coupled receptors remains poorly understood. We previously reported that prostaglandin E receptor EP1 facilitates dopamine D1 receptor signaling in striatal slices and promotes behavioral responses induced by D1 receptor agonists. Here, using human embryonic kidney (HEK)-293T cells expressing D1 and EP1, we have analyzed the mechanism underlying EP1-mediated facilitation of D1 receptor signaling. Fluorescent immunostaining showed that EP1 and D1 receptors are partly colocalized in the cells, and coprecipitation experiments revealed a molecular complex of EP1 and D1 receptors. Treatment of the cells with 17S,17,20-dimethyl-2,5-ethano-6-oxo-PGE1 (ONO-DI-004), an EP1-selective agonist, enhanced cAMP production induced by D1 agonists (±)-6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide (SKF-81297) and 6-chloro-2,3,4,5-tetrahydro-1-(3-methylphenyl)-3-(2-propenyl)-1H-3-benzazepine-7,8-diol hydrobromide (SKF-83822). Although this facilitative effect of EP1 stimulation was not affected by pharmacologic blockade of EP1-induced Ca²âº increase, it was blocked by overexpression of G(tα) as a G(ßγ) scavenger. Consistently, depletion of adenylyl cyclase (AC) 7, a G(ßγ)-sensitive AC isoform, abolished the facilitative action of EP1 on D1-induced cAMP production. Notably, neither G(tα) overexpression nor AC7 depletion affected cAMP production induced by D1 stimulation alone. In contrast, depletion of AC6, another AC isoform, reduced cAMP production induced by D1 stimulation alone, but spared its facilitation by EP1 stimulation. Collectively, these data suggest that, through complex formation with D1, EP1 signaling directs the D1 receptor through G(ßγ) to be coupled to AC7, an AC isoform distinct from those used by the D1 receptor alone, in HEK-293T cells.

Prostaglandin E(2) regulates murine hematopoietic stem/progenitor cells directly via EP4 receptor and indirectly through mesenchymal progenitor cells.

Prostaglandin E(2) (PGE(2)) regulates hematopoietic stem/progenitor cell (HSPC) activity. However, the receptor(s) responsible for PGE(2) signaling remains unclear. Here, we identified EP4 as a receptor activated by PGE(2) to regulate HSPCs. Knockdown of Ep4 in HSPCs reduced long-term reconstitution capacity, whereas an EP4-selective agonist induced phosphorylation of GSK3ß and ß-catenin and enhanced long-term reconstitution capacity. Next, we analyzed the niche-mediated effect of PGE(2) in HSPC regulation. Bone marrow mesenchymal progenitor cells (MPCs) expressed EP receptors, and stimulation of MPCs with PGE(2) significantly increased their ability to support HSPC colony formation. Among the EP receptor agonists, only an EP4 agonist facilitated the formation of HSPC colonies after the coculture with MPCs. PGE(2) up-regulated the expression of cytokine-, cell adhesion-, extracellular matrix-, and protease-related genes in MPCs. We also examined the function of PGE(2)/EP4 signaling in the recovery of the HSPCs after myelosuppression. The administration of PGE(2) or an EP4 agonist facilitated the recovery of HSPCs from 5-fluorouracil (5-FU)-induced myelosuppression, indicating a role for PGE(2)/EP4 signaling in this process. Altogether, these data suggest that EP4 is a key receptor for PGE(2)-mediated direct and indirect regulation of HSPCs.

Orally administered rubiscolin-6, a δ opioid peptide derived from Rubisco, stimulates food intake via leptomeningeal lipocallin-type prostaglandin D synthase in mice.

SCOPE: We found that rubiscolin-6, a δ opioid agonist peptide derived from d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a major protein of green leaves, stimulates food intake after oral administration in mice. We therefore investigated its mechanism. METHODS AND RESULTS: Orexigenic activity after oral administration of rubiscolin-6 was blocked by central administration of naltrindole, an antagonist for δ opioid receptor, suggesting that orally administered rubiscolin-6 stimulates food intake via central δ opioid receptor activation. The orexigenic activity of rubiscolin-6 was inhibited by celecoxib, a cyclooxygenase (COX)-2 inhibitor. The hypothalamic mRNA expression of COX-2 and lipocallin-type (L) prostaglandin D synthase (PGDS) was elevated in response to rubiscolin-6 administration. Rubiscolin-6 stimulated food intake in wild-type and hematopoietic (H)-PGDS knockout (KO), but not L-PGDS KO mice. Interestingly, rubiscolin-6 stimulated food intake in L-PGDS(flox) /Nescre mice, which were deficient in L-PGDS in the brain parenchyma, but not leptomeninges. The orexigenic effect of rubiscolin-6 was abolished by genetic deletion of DP(1) receptor for PGD(2) , and by MK0524 or BIBO3304, an antagonist of DP(1) receptor or of Y(1) receptor for neuropeptide Y, respectively. CONCLUSION: Orally administered rubiscolin-6 may stimulate food intake through COX-2 and leptomeningeal L-PGDS, followed by DP(1) and Y(1) receptors, downstream of the central δ opioid receptor.

A role for mDia, a Rho-regulated actin nucleator, in tangential migration of interneuron precursors.

In brain development, distinct types of migration, radial migration and tangential migration, are shown by excitatory and inhibitory neurons, respectively. Whether these two types of migration operate by similar cellular mechanisms remains unclear. We examined neuronal migration in mice deficient in mDia1 (also known as Diap1) and mDia3 (also known as Diap2), which encode the Rho-regulated actin nucleators mammalian diaphanous homolog 1 (mDia1) and mDia3. mDia deficiency impaired tangential migration of cortical and olfactory inhibitory interneurons, whereas radial migration and consequent layer formation of cortical excitatory neurons were unaffected. mDia-deficient neuroblasts exhibited reduced separation of the centrosome from the nucleus and retarded nuclear translocation. Concomitantly, anterograde F-actin movement and F-actin condensation at the rear, which occur during centrosomal and nuclear movement of wild-type cells, respectively, were impaired in mDia-deficient neuroblasts. Blockade of Rho-associated protein kinase (ROCK), which regulates myosin II, also impaired nuclear translocation. These results suggest that Rho signaling via mDia and ROCK critically regulates nuclear translocation through F-actin dynamics in tangential migration, whereas this mechanism is dispensable in radial migration.

Deficiency of mDia, an actin nucleator, disrupts integrity of neuroepithelium and causes periventricular dysplasia.

During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.

Central control of fever and female body temperature by RANKL/RANK.

Receptor-activator of NF-kappaB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1beta and TNFalpha. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE(2)/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rank(floxed) mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation.

Impaired adrenocorticotropic hormone response to bacterial endotoxin in mice deficient in prostaglandin E receptor EP1 and EP3 subtypes.

Sickness evokes various neural responses, one of which is activation of the hypothalamo-pituitary-adrenal (HPA) axis. This response can be induced experimentally by injection of bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1. Although prostaglandins (PGs) long have been implicated in LPS-induced HPA axis activation, the mechanism downstream of PGs remained unsettled. By using mice lacking each of the four PGE receptors (EP1-EP4) and an EP1-selective antagonist, ONO-8713, we showed that both EP1 and EP3 are required for adrenocorticotropic hormone release in response to LPS. Analysis of c-Fos expression as a marker for neuronal activity indicated that both EP1 and EP3 contribute to activation of neurons in the paraventricular nucleus of the hypothalamus (PVN). This analysis also revealed that EP1, but not EP3, is involved in LPS-induced activation of the central nucleus of the amygdala. EP1 immunostaining in the PVN revealed its localization at synapses on corticotropin-releasing hormone-containing neurons. These findings suggest that EP1- and EP3-mediated neuronal pathways converge at corticotropin-releasing hormone-containing neurons in the PVN to induce HPA axis activation upon sickness.

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts.

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.