Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.

SAMHD1 restricts the deoxyguanosine triphosphate pool contributing to telomere stability in telomerase-positive cells.

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination.

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11ß splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11ßki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11ßki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11ß and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

The displacement of frataxin from the mitochondrial cristae correlates with abnormal respiratory supercomplexes formation and bioenergetic defects in cells of Friedreich ataxia patients.

Friedreich ataxia (FRDA) is a neurodegenerative disease resulting from a severe decrease of frataxin (FXN). Most patients carry a GAA repeat expansion in both alleles of the FXN gene, whereas a small fraction of them are compound heterozygous for the expansion and a point mutation in the other allele. FXN is involved in the mitochondrial biogenesis of the FeS-clusters. Distinctive feature of FRDA patient cells is an impaired cellular respiration, likely due to a deficit of key redox cofactors working as electrons shuttles through the respiratory chain. However, a definite relationship between FXN levels, FeS-clusters assembly dysregulation and bioenergetics failure has not been established. In this work, we performed a comparative analysis of the mitochondrial phenotype of cell lines from FRDA patients, either homozygous for the expansion or compound heterozygotes for the G130V mutation. We found that, in healthy cells, FXN and two key proteins of the FeS-cluster assembly machinery are enriched in mitochondrial cristae, the dynamic subcompartment housing the respiratory chain. On the contrary, FXN widely redistributes to the matrix in FRDA cells with defects in respiratory supercomplexes assembly and altered respiratory function. We propose that this could be relevant for the early mitochondrial defects afflicting FRDA cells and that perturbation of mitochondrial morphodynamics could in turn be critical in terms of disease mechanisms.

The Interplay between Ghrelin and Microglia in Neuroinflammation: Implications for Obesity and Neurodegenerative Diseases.

Numerous studies have shown that microglia are capable of producing a wide range of chemokines to promote inflammatory processes within the central nervous system (CNS). These cells share many phenotypical and functional characteristics with macrophages, suggesting that microglia participate in innate immune responses in the brain. Neuroinflammation induces neurometabolic alterations and increases in energy consumption. Microglia may constitute an important therapeutic target in neuroinflammation. Recent research has attempted to clarify the role of Ghre signaling in microglia on the regulation of energy balance, obesity, neuroinflammation and the occurrence of neurodegenerative diseases. These studies strongly suggest that Ghre modulates microglia activity and thus affects the pathophysiology of neurodegenerative diseases. This review aims to summarize what is known from the current literature on the way in which Ghre modulates microglial activity during neuroinflammation and their impact on neurometabolic alterations in neurodegenerative diseases. Understanding the role of Ghre in microglial activation/inhibition regulation could provide promising strategies for downregulating neuroinflammation and consequently for diminishing negative neurological outcomes.

Ghrelin peptide improves glial conditioned medium effects on neuronal differentiation of human adipose mesenchymal stem cells.

The influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.

SAMHD1-deficient fibroblasts from Aicardi-Goutières Syndrome patients can escape senescence and accumulate mutations.

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

Separase prevents genomic instability by controlling replication fork speed.

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis.

Common fragile site instability in normal cells: Lessons and perspectives.

Mechanisms and events related to common fragile site (CFS) instability are well known in cancer cells. Here, we argue that normal cells remain an important experimental model to address questions related to CFS instability in the absence of alterations in cell cycle and DNA damage repair pathways, which are common features acquired in cancer. Furthermore, a major gap of knowledge concerns the stability of CFSs during gametogenesis. CFS instability in meiotic or postmeiotic stages of the germ cell line could generate chromosome deletions or large rearrangements. This in turn can lead to the functional loss of the several CFS-associated genes with tumor suppressor function. Our hypothesis is that such mutations can potentially result in genetic predisposition to develop cancer. Indirect evidence for CFS instability in human germ cells has been provided by genomic investigations in family pedigrees associated with genetic disease. The issue of CFS instability in the germ cell line should represent one of the future efforts, and may take advantage of the existence of sequence and functional conservation of CFSs between rodents and humans.

The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion.

It is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich's ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat.

Rad54/Rad54B deficiency is associated to increased chromosome breakage in mouse spermatocytes.

Rad54 protein is a key player of the homologous recombination pathway, required for deposition and stabilisation of Rad51 foci at double strand breaks. Its role at the meiotic prophase, when double strand breaks are physiologically introduced to allow recombination, is well described. However, the hypothesis that Rad54 deficiency affect chromosome integrity of germ cells in unirradiated and irradiated animals has not been tested yet. In this study, the occurrence of spontaneous and X-ray-induced chromosome aberrations was assessed by analysis of spermatocyte MI spreads or by application of micronucleus assay in early spermatids isolated from wild type and Rad54/Rad54B knockout (KO) mice. In Rad54/Rad54B KO mice, the spontaneous chromosome aberration frequency detected at MI was >10-fold higher than in wild type animals. In addition, after exposure to 1 Gy X-rays at the radiosensitive stage of diplotene, an enhanced response to radiation was observed in Rad54-deficient animals, corresponding to a 2-3 sensitivity factor in comparison to wild type mice. Also the spontaneous frequency of micronucleated round spermatids was on the average 10-fold higher in KO than in wild type mice, indicating that Rad54/Rad54B KO spermatocytes carrying chromosome aberrations are able to pass through the meiotic divisions and to continue the spermatogenesis process. Our results provide the first evidence of the role of Rad54/Rad54B in the maintenance of a stable karyotype during male meiosis, and suggest that Rad54/Rad54B deficiency may impact on the DNA integrity of developing mouse gametes.

Ghrelin-containing neurons in the olfactory bulb send collateralized projections into medial amygdaloid and arcuate hypothalamic nuclei: neuroanatomical study.

Ghrelin, a gastrointestinal hormone, is a modulator of the sense of smell. The main source of ghrelin in the central nervous system has been mainly observed in specific populations of hypothalamic neurons. An increasing number of studies have reported ghrelin synthesis and its effect on neurons outside the hypothalamus. Ghrelin and its receptors are expressed in the olfactory bulbs and in other centres of the brain, such as the amygdala, for processing olfactory signals, pyramidal neurons of the cerebral cortex and the dorsal vagal complex of the medulla oblongata. It is known that ghrelin is involved in cognitive mechanisms and eating behaviours, in fact, its expression increases in anticipation of food intake. In order to identify the existence of centrifugal direct afferents from the main olfactory bulb to the medial amygdala and the hypothalamus arcuate nucleus, in this work we used two retrograde tracers, Dil and Fluoro Gold, and immunohistochemical procedure to visualize positive ghrelin neurons. Our paper provides neuroanatomic support for the ghrelin modulation of smell. Our results show that ghrelin neuron projections from mitral cells of bulbs can transmit olfactory information via branching connections to the amygdala and the hypothalamus. This pathway could play an important role in regulating feeding behaviour in response to odours.

One-Pot Conversion of Epoxidized Soybean Oil (ESO) into Soy-Based Polyurethanes by MoCl2O2 Catalysis.

An innovative and eco-friendly one-pot synthesis of bio-based polyurethanes is proposed via the epoxy-ring opening of epoxidized soybean oil (ESO) with methanol, followed by the reaction of methoxy bio-polyols intermediates with 2,6-tolyl-diisocyanate (TDI). Both synthetic steps, methanolysis and polyurethane linkage formation, are promoted by a unique catalyst, molybdenum(VI) dichloride dioxide (MoCl2O2), which makes this procedure an efficient, cost-effective, and environmentally safer method amenable to industrial scale-up.

Chromosome Imbalances in Cancer: Molecular Cytogenetics Meets Genomics.

Genomic instability is a hallmark of cancer, and it is well-known that in several cancers the karyotype is unstable and rapidly evolving. Molecular cytogenetics has contributed to the description and interpretation of cancer karyotypes, in particular through multicolor FISH approaches which can define even complex chromosome rearrangements. The introduction of genome-wide methods has made available a powerful set of tools with higher resolution than cytogenetics, thus appropriate to comprehend the huge variability of cancer cells. This review focuses on novel findings deriving from the combination of cytogenetic and genomic approaches in cancer research.

Evidence for a trigeminal mesencephalic-hypoglossal nuclei loop involved in controlling vibrissae movements in the rat.

Previous studies performed in rats showed that the whisker-pad motor innervation involves not only the facial nerve, but also some hypoglossal neurons whose axons travel within the trigeminal infraorbital nerve (ION) and target the extrinsic muscles surrounding the whisker-pad macrovibrissae. Furthermore, the electrical stimulation of the ION induced an increase in the EMG activity of these muscles, while the hypoglossal nucleus stimulation elicited evoked potentials and single motor unit responses. However, the existence of a neural network able to involve the XIIth nucleus in macrovibrissae whisking control was totally unknown until now. Since other recent experiments demonstrated that: (1) the mesencephalic trigeminal nucleus (Me5) neurons respond to both spontaneous and artificial movements of macrovibrissae, and (2) the Me5 peripheral terminals provide a monosynaptic sensory innervation to the macrovibrissae, the present study was aimed at analyzing a possible role of the Me5 nucleus as a relay station in the sensory-motor loop that involves the XIIth nucleus neurons in rhythmic whisking control. Two tracers were used in the same animal: Fluoro Gold, which was injected into the whisker pad to retrogradely label the hypoglossal whisker-pad projection neurons, and Dil, which was instead injected into the Me5 to label its projections to these hypoglossal neurons. Results demonstrated that terminals of the Me5 neurons monosynaptically target the hypoglossal whisker-pad projection neurons. The functional role of this sensory-motor connection is discussed, with particular regard to a hypothesized proprioceptive reflex in whisker-pad extrinsic muscles that can be elicited by the activation of the Me5 macrovibrissae receptors.

1,25(OH)2 vitamin D3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice.

Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbß3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.

Serum- and glucocorticoid-inducible kinase 1 sensitive NF-κB signaling in dendritic cells.

BACKGROUND/AIMS: Dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity, are required for initiation of specific T cell-driven immune responses. Phosphoinositide-3-kinase (PI3K) suppresses proinflammatory cytokine production in DCs, which limits T helper (Th1) polarization. PI3K is in part effective by downregulation of transcription factor NF-κB. Downstream signaling elements of PI3K include serum- and glucocorticoid-inducible kinase 1 (SGK1) and its phosphorylation target N-myc downstream regulated gene 1 (NDRG1). The present study explored whether SGK1 and NDRG1 play a role in the regulation of NF-κB and DC-maturation. METHODS: DCs were isolated from bone marrow (BMDCs) or spleen of mice lacking functional SGK1 (sgk1(-/-)) and corresponding wild type mice (sgk1(+/+)). Protein abundance was determined by Western blotting. Transcription was inhibited by siRNA. Abundance of maturation markers was quantified by flow cytometry. FITC-dextran uptake was determined to quantify phagocytosis. RESULTS: NDRG1 was similarly expressed in sgk1(+/+) and sgk1(-/-)BMDCs, but SGK1-dependent phosphorylation of NDRG-1 was decreased in sgk1(-/-)BMDCs. Silencing of NDRG1 in sgk1(+/+)BMDCs as compared to control empty vector-treated BMDCs enhanced nuclear abundance of NF-κB subunit p65. Moreover, the abundance of phosphorylated NF-κB inhibitor IκBα, of phosphorylated IκB kinase (IKKα/ß) and of nuclear p65 were significantly higher in sgk1(-/-)BMDCs than in sgk1(+/+)BMDCs. Expression of maturation markers, MHC II, and CD86, was significantly larger and phagocytic capacity was significantly lower in sgk1(-/-) than in sgk1(+/+)BMDCs. Expression of CD86 and MHCII was also significantly higher in DCs isolated from the spleen of sgk1(-/-) mice than those from sgk1(+/+)mice. CONCLUSION: SGK1 and NDRG1 participate in the regulation of NF-κB signaling in and maturation of DCs.

Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.

BACKGROUND/AIMS: The protein kinase Akt2/PKBß is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling. METHODS: DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. RESULTS: Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration. CONCLUSION: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Chorein sensitivity of cytoskeletal organization and degranulation of platelets.

Chorea-acanthocytosis (ChAc), a lethal disease caused by defective chorein, is characterized by neurodegeneration and erythrocyte acanthocytosis. The functional significance of chorein in other cell types remained ill-defined. The present study revealed chorein expression in blood platelets. As compared to platelets from healthy volunteers, platelets from patients with ChAc displayed a 47% increased globular/filamentous actin ratio, indicating actin depolymerization. Moreover, phosphoinositide-3-kinase subunit p85 phosphorylation, p21 protein-activated kinase (PAK1) phosphorylation, as well as vesicle-associated membrane protein 8 (VAMP8) expression were significantly reduced in platelets from patients with ChAc (by 17, 22, and 39%, respectively) and in megakaryocytic (MEG-01) cells following chorein silencing (by 16, 54, and 11%, respectively). Activation-induced platelet secretion from dense granules (ATP release) and α granules (P-selectin exposure) were significantly less (by 55% after stimulation with 1 µg/ml CRP and by 33% after stimulation with 5 µM TRAP, respectively) in ChAc platelets than in control platelets. Furthermore, platelet aggregation following stimulation with different platelet agonists was significantly impaired. These observations reveal a completely novel function of chorein, i.e., regulation of secretion and aggregation of blood platelets.