Physiological Downregulation Research Articles

Photoacclimation by Arctic cryoconite phototrophs.

Cryoconite is a matrix of sediment, biogenic polymer and a microbial community that resides on glacier surfaces. The phototrophic component of this community is well adapted to this extreme environment, including high light stress. Photoacclimation of the cryoconite phototrophic community on Longyearbreen, Svalbard, was investigated using in situ variable chlorophyll fluorescence. Rapid light curves (RLCs) and induction-recovery curves were used to analyse photosystem II quantum efficiency, relative electron transport rate and forms of downregulation including non-photochemical quenching (NPQ) and state transitions in cyanobacteria. Phototrophs used a combination of behavioural and physiological photochemical downregulation. Behavioural downregulation is hypothesised to incorporate chloroplast movement and cell or filament positioning within the sediment matrix in order to shade from high light, which resulted in a lack of saturation of RLCs and hence overestimation of productivity. Physiological downregulation likely consisted of biphasic NPQ, comprising a steadily induced light-dependent form and a light-independent NPQ that was not reversed with decreasing light intensity. State transitions by cyanobacteria were the most likely physiological downregulation employed by cyanobacteria within the mixed phototroph community. These findings demonstrate that cryoconite phototrophs combine multiple forms of physiological and behavioural downregulation to optimise light exposure and maximise photosynthetic productivity. This plasticity of photoacclimation enables them to survive productively in the high-light stress environment on the ice surface.

Synaptotagmin-2, and -1, linked to neurotransmission impairment and vulnerability in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the most frequent genetic cause of infant mortality. The disease is characterized by progressive muscle weakness and paralysis of axial and proximal limb muscles. It is caused by homozygous loss or mutation of the SMN1 gene, which codes for the Survival Motor Neuron (SMN) protein. In mouse models of the disease, neurotransmitter release is greatly impaired, but the molecular mechanisms of the synaptic dysfunction and the basis of the selective muscle vulnerability are unknown. In the present study, we investigated these open questions by comparing the molecular and functional properties of nerve terminals in severely and mildly affected muscles in the SMNΔ7 mouse model. We discovered that synaptotagmin-1 (Syt1) was developmentally downregulated in nerve terminals of highly affected muscles but not in low vulnerable muscles. Additionally, the expression levels of synaptotagmin-2 (Syt2), and its interacting protein, synaptic vesicle protein 2 (SV2) B, were reduced in proportion to the degree of muscle vulnerability while other synaptic proteins, such as syntaxin-1B (Stx1B) and synaptotagmin-7 (Syt7), were not affected. Consistently with the extremely low levels of both Syt-isoforms, and SV2B, in most affected neuromuscular synapses, the functional analysis of neurotransmission revealed highly reduced evoked release, altered short-term plasticity, low release probability, and inability to modulate normally the number of functional release sites. Together, we propose that the strong reduction of Syt2 and SV2B are key factors of the functional synaptic alteration and that the physiological downregulation of Syt1 plays a determinant role in muscle vulnerability in SMA.

FBXO11 inactivation leads to abnormal germinal-center formation and lymphoproliferative disease

AbstractThe BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)–mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor–mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice.

Combining Sun-Induced Chlorophyll Fluorescence and Photochemical Reflectance Index Improves Diurnal Modeling of Gross Primary Productivity

Sun-induced chlorophyll fluorescence (F) is a novel remote sensing parameter providing an estimate of actual photosynthetic rates. A combination of this new observable and Monteith’s light use efficiency (LUE) concept was suggested for an advanced modeling of gross primary productivity (GPP). In this demonstration study, we evaluate the potential of both F and the more commonly used photochemical reflectance index (PRI) to approximate the LUE term in Monteith’s equation and eventually improve the forward modeling of GPP diurnals. Both F and the PRI were derived from ground and airborne based spectrometer measurements over two different crops. We demonstrate that approximating dynamic changes of LUE using F and PRI significantly improves the forward modeling of GPP diurnals. Especially in sugar beet, a changing photosynthetic efficiency during the day was traceable with F and incorporating F in the forward modeling significantly improved the estimation of GPP. Airborne data were projected to produce F and PRI maps for winter wheat and sugar beet fields over the course of one day. We detected a significant variability of both, F and the PRI within one field and particularly between fields. The variability of F and PRI was higher in sugar beet, which also showed a physiological down-regulation of leaf photosynthesis. Our results underline the potential of F to serve as a superior indicator for the actual efficiency of the photosynthetic machinery, which is linked to physiological responses of vegetation.

Abstract 377: Phorbol 12-myristate 13-acetate Downregulates Tissue Factor Gene Expression in Human Pericytes

Tissue factor (TF) is a transmembrane receptor for factor VII/VIIa that plays a central role in hemostasis and angiogenesis. While TF is generally upregulated in pathologic conditions, we have reported that TF is downregulated in pericytes around vessels near a healing cutaneous wound. As the first demonstration of physiologic downregulation of TF, this finding suggested a potential means of modulating TF expression for therapeutic purposes. The goal of this study was to contribute to implementation of future therapies by elucidating mechanisms that regulate loss pericyte TF. To identify mediators of TF downregulation we utilized primary cultures of human placental pericytes to screen growth factors involved in wound healing including TGFβ, bFGF, VEGF, PDGF and ANG2. None significantly reduced pericyte TF expression as assessed by western blotting. We next tested agents that directly activate signaling pathways. Phorbol 12-myristate 13-acetate (PMA) triggered ~60% reduction in TF protein 4 hours after treatment. Complete loss of TF occurred by 8 hours (p<0.001) and remained until the experiment was terminated 24 hours after PMA addition. These results suggested that TF loss is mediated, at least in part, by downregulation of TF gene expression. We utilized qRT-PCR to determine the effects of PMA on synthesis of TF transcripts 4, 8, 12, and 24 hours after treatment. TF mRNA levels were unchanged 4 hours after treatment. However, TF transcripts decreased 4 and 6 fold 8 hours and 12 hours after PMA, respectively (p<0.01). 24 hours after treatment the amount of TF mRNA rebounded, yet remained 2 fold lower than the control (p<0.05). To determine if the decrease in TF transcripts is caused by mRNA destabilization, pericytes were treated with actinomycin D prior to PMA. Degradation of TF transcripts in PMA-treated cells was similar to that of DMSO-treated control cells at all time points (p=ns), indicating that PMA-mediated downregulation of TF expression occurs primarily through inhibition of mRNA synthesis rather than through destabilization of existing transcripts. In conclusion, our results demonstrate a role for transcriptional downregulation in PMA-mediated loss of pericyte TF protein, findings that pave the way for future in vivo studies of wound healing.

Is Adrenal Insufficiency a Rare Disease?

Adrenal insufficiency (AI) is a potentially life-threatening condition and it is of utmost importance to identify and adequately manage affected individuals. Diagnosis is often delayed, probably partly because diseases of the adrenal or pituitary region that cause primary AI (PAI) or central AI are relatively rare conditions. However, iatrogenic AI, i.e. the physiological downregulation of the hypothalamic-pituitary-adrenal axis and adrenal atrophy caused by glucocorticoid treatment for different inflammatory conditions is likely to be considerably more common. The type of glucocorticoid, dose and duration of treatment are factors to consider when trying to predict the risk of developing symptoms of AI. However, the considerable individual variation in the sensitivity for developing iatrogenic AI impedes prediction. In industrialized countries, autoimmune adrenalitis accounts for the majority of cases of PAI. Among children, genetic conditions - in particular congenital adrenal hyperplasia - need to be considered. Important risk groups for central AI are patients with tumours in the hypothalamic-pituitary region, moderate-to-severe traumatic head injury and patients who receive cranial radiotherapy or cytotoxic T-lymphocyte antigen 4 blockade treatment. Structured endocrine follow-up is essential in these groups. Health workers need to be attentive to these potentially fatal conditions and at-risk populations should be carefully informed about symptoms and signs of AI.

Downregulation of Tissue Factor in Pericytes By a Protein Kinase C-Dependent Mechanism

Tissue factor (TF) is a transmembrane protein in the type II cytokine receptor family that serves as a cofactor for factor VIIa activity, and is essential for normal initiation of hemostasis. Its expression is generally upregulated in the settings of inflammation, immune activation, tissue injury and malignancy. TF has biological functions in addition to its role hemostasis. In malignancy, high expression of TF by tumor cells promotes local angiogenesis. By contrast, we have reported that expression of perivascular TF is downregulated within 24 hours after wounding, and remains low around angiogenic vessels during physiologic wound healing. The mechanism(s) of physiologic downregulation of TF, and its role in physiologic angiogenesis have not been previously characterized. Therefore, the objective of this study was to identify mechanisms contributing to TF downregulation in primary human pericytes. We determined that loss of TF in human pericytes can be recapitulated in vitro by treating pericyte cultures with phorbol 12-myristate 13-acetate (PMA). The time course and degree of TF downregulation over 24 hours, as shown in the figure, is similar to that observed within the first 24 hours in vivo. Since PMA is a potent activator of Protein Kinase C (PKC), we determined the role of PKC in PMA-mediated TF loss by pretreating pericytes with the broad spectrum PKC inhibitor, Go 6983, prior to stimulation with PMA. PKC inhibition eliminated PMA-induced TF loss, supporting the conclusion that the PMA effect occurs through a PKC-dependent mechanism. PKCa has been shown to induce phosphorylation of the TF cytoplasmic tail, which results in budding of TF-rich microvesicles from the plasma membrane. PMA also triggers activation of the cell-surface protease ADAM17. Based on domain homology with other targets, ADAM17 could potentially cleave and release the extracellular domain of TF from the cell surface. To determine if TF loss occurs through its release from the cell by either mechanism we utilized biotin labeling of surface TF, streptavidin pull down, and western blot analysis to assess trafficking of TF into the culture media. Our results show that surface TF significantly decreases within 2 hours of PMA treatment. However, we failed to detect biotinylated TF in the culture media. Isolation of microvesicles from the media followed by western blotting for TF showed that pericytes do release TF-associated microvesicles at a basal rate, but this rate is not affected by PMA. Taken together, these data show that PMA induces downregulation of TF in primary human pericytes through a PKC-dependent mechanism, and that this mechanism does not stimulate release of TF from the cell in either microvesicles or by proteolytic cleavage of the TF extracellular domain. Our working hypothesis is that PMA triggers internalization of surface TF via activation of protein kinase C, leading to its subsequent degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation.

Interferon-γ (IFN-γ) primes macrophages for enhanced inflammatory activation by Toll-like receptors (TLRs) and microbial killing, but little is known about the regulation of cell metabolism or mRNA translation during priming. We found that IFN-γ regulates human macrophage metabolism and translation by targeting the kinases mTORC1 and MNK that both converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages revealed that IFN-γ selectively modulates the macrophage translatome to promote inflammation, further reprogram metabolic pathways, and modulate protein synthesis. These results add IFN-γ-mediated metabolic reprogramming and translational regulation as key components of classical inflammatory macrophage activation.

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

The A2B adenosine receptor (A2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A2B AR in the control of osteoblast differentiation, bone formation and fracture repair.

Temporal Lineage Tracing of Aire-Expressing Cells Reveals a Requirement for Aire in Their Maturation Program

Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential for gaining insight into the roles of Aire in establishment of self-tolerance. In this study, we monitored the maturation program of AEL-mTECs by temporal lineage tracing, in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under control of the Aire regulatory element were crossed with reporter strains. We estimated that the half-life of AEL-mTECs subsequent to Aire expression was ∼7-8 d, which was much longer than that reported previously, owing to the existence of a post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in medullary thymic epithelial cells (mTECs) might result in their apoptosis for efficient cross-presentation of self-antigens expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs, as exemplified by the lack of physiological downregulation of CD80 during the post-Aire stage in Aire-deficient mice, thus accounting for the abnormally increased CD80(high) mTECs seen in such mice. Of interest, increased CD80(high) mTECs in Aire-deficient mice were not mTEC autonomous and were dependent on cross-talk with thymocytes. These results further support the roles of Aire in the differentiation program of AEL-mTECs.

Anti-Müllerian hormone (AMH) downregulation in late antral stages is impaired in PCOS patients. A study in normo-ovulatory and PCOS patients undergoing in vitro maturation (IVM) treatments

Purpose: To prospectively study the AMH expression and secretion pattern in mural granulosa cells (GCs) and follicular fluid (FF) from small follicles and medium follicles that were collected from normo-ovulatory (NO) and polycystic ovary syndrome (PCOS) patients undergoing in vitro maturation (IVM) treatments.Methods: FF AMH levels and mRNA expression of mural GCs were measured in small (≤10 mm) and medium size follicles (11–15 mm) obtained from IVM treatments and large size follicles (≥16 mm) obtained from in vitro fertilization treatments.Results: First, we show that AMH expression and protein level in the FF of NO patients were significantly higher in the small size follicles than in the medium and large size follicles (p < 0.003). We could not demonstrate these differences in PCOS patients. Second, we found significantly higher levels of AMH protein and mRNA in the large and medium (but not small) size follicles of PCOS patients compared to follicles from NO patients (p < 0.02). Finally, we observed a positive correlation between FF AMH of small and medium size follicles from NO patients and serum AMH (p < 0.03 and p < 0.0002, respectively).Conclusions: Our data demonstrate a pathological dysregulation of AMH expression and secretion in follicles from PCOS patients and emphasize the association between the physiological downregulation of AMH and follicular antral health.

Temporal lineage tracing of Aire-expressing cells reveals a requirement of Aire for their maturation program

Event Abstract Back to Event Temporal lineage tracing of Aire-expressing cells reveals a requirement of Aire for their maturation program Mitsuru Matsumoto1* 1 Institute for Enzyme Research, University of Tokushima, Division of Molecular Immunology, Japan Understanding the cellular dynamics of Aire-expressing lineage(s) among medullary thymic epithelial cells (AEL-mTECs) is essential to gain insight into the roles of Aire for establishing self-tolerance. We sought to monitor the maturation program of AEL- mTECs by a temporal lineage tracing in which bacterial artificial chromosome transgenic mice expressing tamoxifen-inducible Cre recombinase under the control of the Aire regulatory element were crossed with reporter strains. We estimated the half-life of AEL- mTECs subsequent to Aire expression approximately 7~8 days, which was much longer than previously reported due to the existence of post-Aire stage. We found that loss of Aire did not alter the overall lifespan of AEL-mTECs, inconsistent with the previous notion that Aire expression in mTECs might result in their apoptosis for efficient cross presentation of self-Ags expressed by AEL-mTECs. In contrast, Aire was required for the full maturation program of AEL-mTECs as exemplified by the lack of physiological down-regulation of CD80 during the post-Aire stage in Aire-deficient mice, which accounts for the increased CD80high mTECs abnormally seen in Aire-deficient mice. The results further enforce the roles of Aire in the differentiation program of AEL-mTECs. Keywords: AIRE, thymic epithelial cells, Autoimmune Diseases, tolerance mechanisms, fate mapping Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Matsumoto M (2013). Temporal lineage tracing of Aire-expressing cells reveals a requirement of Aire for their maturation program. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00617 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Mitsuru Matsumoto, Institute for Enzyme Research, University of Tokushima, Division of Molecular Immunology, Tokushima, Tokushima, 770-8503, Japan, mitsuru@ier.tokushima-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mitsuru Matsumoto Google Mitsuru Matsumoto Google Scholar Mitsuru Matsumoto PubMed Mitsuru Matsumoto Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Identification of soluble CD146 as a regulator of trophoblast migration: potential role in placental vascular development

Both vasculogenesis and angiogenesis occur during normal placental vascular development. Additionally, the placenta undergoes a process of vascular mimicry (pseudo-vasculogenesis) where the placental extravillous trophoblast (EVT) that invade the spiral arteries convert from an epithelial to an endothelial phenotype during normal pregnancy. As soluble CD146 (sCD146) constitutes a new physiological factor with angiogenic properties, we hypothesized that it could be involved in the regulation of placental vascular development by acting on EVT. Using placental villous explants, we demonstrated that sCD146 inhibits EVT outgrowth. Consistently, we showed that sCD146 inhibits the ability of EVT cells (HTR8/SVneo) to migrate, invade and form tubes in Matrigel, without affecting their proliferation or apoptosis. The involvement of sCD146 in human pregnancy was investigated by evaluation of sCD146 levels in 50 pregnant women. We observed physiological down-regulation of sCD146 throughout pregnancy. These results prompted us to investigate the effect of prolonged sCD146 administration in a rat model of pregnancy. Repeated systemic sCD146 injections after coupling caused a significant decrease of pregnancy rate and number of embryos. Histological studies performed on placenta evidenced a reduced migration of glycogen cells (analogous to EVT in rat) in sCD146-treated rats. We propose that in human, sCD146 could represent both an attractive biomarker of placental vascular development and a therapeutic target in pregnancy complications associated with pathological angiogenesis.

Analysis of Pacific oyster larval proteome and its response to high-CO2

Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO2 due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO2. Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae.

The Sedoheptulose Kinase CARKL Directs Macrophage Polarization through Control of Glucose Metabolism

SummaryImmune cells are somewhat unique in that activation responses can alter quantitative phenotypes upwards of 100,000-fold. To date little is known about the metabolic adaptations necessary to mount such dramatic phenotypic shifts. Screening for novel regulators of macrophage activation, we found nonprotein kinases of glucose metabolism among the most enriched classes of candidate immune modulators. We find that one of these, the carbohydrate kinase-like protein CARKL, is rapidly downregulated in vitro and in vivo upon LPS stimulation in both mice and humans. Interestingly, CARKL catalyzes an orphan reaction in the pentose phosphate pathway, refocusing cellular metabolism to a high-redox state upon physiological or artificial downregulation. We find that CARKL-dependent metabolic reprogramming is required for proper M1- and M2-like macrophage polarization and uncover a rate-limiting requirement for appropriate glucose flux in macrophage polarization.

Moderate Long-Term Modulation of Neuropeptide Y in Hypothalamic Arcuate Nucleus Induces Energy Balance Alterations in Adult Rats

Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons. Hypothalamic NPY levels undergo wide-ranging oscillations during the circadian cycle and in response to fasting and peripheral hormones (from 0.25 to 10-fold change). The aim of the present study was to evaluate the impact of a moderate long-term modulation of NPY within the ARC neurons on food consumption, body weight gain and hypothalamic neuropeptides. We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively. Our work shows that a moderate overexpression of NPY was sufficient to induce diurnal over-feeding, sustained body weight gain and severe obesity in adult rats. Additionally, the circulating levels of leptin were elevated but the immunoreactivity (ir) of ARC neuropeptides was not in accordance (POMC-ir was unchanged and AGRP-ir increased), suggesting a disruption in the ability of ARC neurons to response to peripheral metabolic alterations. Furthermore, a dysfunction in adipocytes phenotype was observed in these obese rats. In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

Down-regulation of stathmin expression is required for megakaryocyte maturation and platelet production

AbstractThe final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

Stathmin-Mediated Dysregulation of Microtubules Impairs Megakaryocyte Maturation and Platelet Production

AbstractAbstract 548Maturation of megakaryocytes (MK) consists of unique processes like polyploidization and proplatelet formation that are critical for efficient platelet production. Both processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton. MT form a complex, spherical mitotic spindle that mediates polyploidization, represent the structural scaffold for proplatelet extension and enable the transport of cytoplasmic organelles into nascent platelets. While the structural organization of the MT cytoskeleton in MK is very well described at the morphological level, the mechanisms responsible for the regulation of MT dynamics during these processes are largely unknown. Stathmin is a highly conserved MT-regulatory protein that modulates MT dynamics by promoting MT depolymerization. Previous studies suggested a potential role for stathmin in MK polyploidization. However, these studies that were performed in chemically-induced transformed cell lines reached contradictory conclusions about the role of stathmin in megakaryopoiesis. We addressed this controversy by utilizing a more physiologically relevant experimental model of megakaryopoiesis in which primary human MK are grown in tissue culture. First, we optimized a MK liquid culture system that allows for efficient generation of mature MK capable of producing platelets in culture. Using this system, we demonstrated that the high levels of stathmin mRNA in proliferating MK progenitors markedly decline with maturation. We used an FIV-based lentiviral expression system to prevent this physiological downregulation of stathmin expression and investigated the effects of sustained expression on MK maturation. The sustained expression of a constitutively active form of stathmin in MK resulted in approximately 2-fold reduction in glycoprotein GPIb expression, 3.5-fold reduction in platelet factor 4 expression and loss of up-regulation of GATA-1 expression which is normally seen during maturation. This demonstrates that the sustained activity of stathmin prevents the up-regulation of three different markers of MK maturation, supporting the hypothesis that physiological downregulation of stathmin is required for proper MK differentiation. We also investigated the effects of sustained stathmin expression on polyploidization by fluorescence in situ hybridization. These studies show that stathmin inhibited the ability of MK to achieve high levels of polyploidy. Finally, we evaluated the effects of sustained stathmin expression on the ability of MK to produce platelets in vitro. We found that the number of platelets derived by MK expressing sustained levels of stathmin was reduced by approximately 50% compared to those produced by control MK. This demonstrates that sustained stathmin expression has a negative effect on platelet production, supporting the importance of MT polymerization in this process. This is the first demonstration that alterations of expression of a MT-regulatory protein can interfere with platelet production by primary MK. In summary, these studies support the importance of MT regulation during MK maturation and suggest that the physiologic downregulation of stathmin expression is biologically important in the processes of MK maturation and platelet production. Very high levels of stathmin expression are frequent in hematological malignancies such as leukemias and myelodysplastic syndrome. Inefficient platelet production is one of the most important features of these malignancies where bleeding from low platelet counts is one of the most common causes of death. Our studies open new avenues of investigation into the mechanisms responsible for MK maturation that may prove relevant for pathological conditions characterized by aberrant stathmin expression and/or function. Disclosures:No relevant conflicts of interest to declare.

Physiological down-regulation and positive emotion in marital interaction.

Previous studies have demonstrated that 1 function of positive emotion is the undoing of physiological arousal produced by negative emotions. These studies have used single-subject paradigms, in which emotions were induced by films in college-age individuals. In the present study, we examined the relationship between physiological down-regulation and positive emotion in a sample of 149 middle-aged and older married couples engaged in a 15-min discussion of an area of marital conflict. During the conversation, autonomic and somatic physiological activity was measured, and emotional behaviors were recorded and subsequently coded. We found that during 20-s periods of down-regulation (where physiology transitioned from high arousal to low arousal), couples showed an increase in positive emotional behavior compared with periods without down-regulation. The finding was quite robust, suggesting that the undoing effect of positive emotion generalizes across age, sex, and marital satisfaction. The advantages of using positive emotion as an emotion regulation strategy are discussed.