Glycogen synthase kinase-3 is essential for β-arrestin-2 complex formation and lithium-sensitive behaviors in mice

Lithium is the first-line therapy for bipolar disorder. However, its therapeutic target remains controversial. Candidates include inositol monophosphatases, glycogen synthase kinase-3 (GSK-3), and a β-arrestin-2/AKT/protein phosphatase 2A (β-arrestin-2/AKT/PP2A) complex that is known to be required for lithium-sensitive behaviors. Defining the direct target(s) is critical for the development of new therapies and for elucidating the molecular pathogenesis of this major psychiatric disorder. Here, we show what we believe to be a new link between GSK-3 and the β-arrestin-2 complex in mice and propose an integrated mechanism that accounts for the effects of lithium on multiple behaviors. GSK-3β (Gsk3b) overexpression reversed behavioral defects observed in lithium-treated mice and similar behaviors observed in Gsk3b+/- mice. Furthermore, immunoprecipitation of striatial tissue from WT mice revealed that lithium disrupted the β-arrestin-2/Akt/PP2A complex by directly inhibiting GSK-3. GSK-3 inhibitors or loss of one copy of the Gsk3b gene reduced β-arrestin-2/Akt/PP2A complex formation in mice, while overexpression of Gsk3b restored complex formation in lithium-treated mice. Thus, GSK-3 regulates the stability of the β-arrestin-2/Akt/PP2A complex, and lithium disrupts the complex through direct inhibition of GSK-3. We believe these findings reveal a new role for GSK-3 within the β-arrestin complex and demonstrate that GSK-3 is a critical target of lithium in mammalian behaviors.     

Inactivation of Wnt/β-catenin signaling in human adipose-derived stem cells is necessary for chondrogenic differentiation and maintenance

The Wnt/β-catenin signaling pathway plays critical roles in self-renewal and differentiation of mesenchymal stem cells. However, very little is known about its role in the chondrogenesis of human adipose-derived stem cells (hADSCs). In this study, we analyzed protein expression of several key components of the Wnt/β-catenin signaling pathway using a 21-day in vitro model of hADSC chondrogenesis. Wnt1, β-catenin, and GSK3β levels increased sharply at day 12, peaked at day 18, and then declined. Expression of TCF1, a target gene of Wnt/β-catenin signaling, closely followed that of Wnt1. These results were consistent with changes in endonuclear β-catenin levels. Gene expression of the chondrocyte-specific markers, collagen type II (COL II), SOX9, and aggrecan, increased during hADSC chondrogenesis, peaked at day 12, and then declined. Adding a Wnt inhibitor (days 0–21) resulted in consistently elevated levels of COL II, SOX9, and aggrecan mRNA. In contrast, adding Wnt1 (days 0–21) to cultures led to sustained Wnt/β-catenin signaling over the 21 days and suppressed expression of chondrocyte-specific markers. Moreover, adding Wnt1 at late stages of differentiation (day 18) further diminished chondrocyte-specific marker expression. Together, these results showed that inactivation of Wnt/β-catenin signaling is needed for the progression of chondrogenesis and the maturation and phenotype maintenance of chondroid cells.     

NF-κB is required for Smac mimetic-mediated sensitization of glioblastoma cells for γ-irradiation-induced apoptosis

Evasion of apoptosis contributes to radioresistance of glioblastoma, calling for novel strategies to overcome apoptosis resistance. In this study, we investigated the potential of the small molecule Smac mimetic BV6 to modulate radiosensitivity of glioblastoma cells. Here, we identify a novel proapoptotic function of NF-κB in γ-irradiation-induced apoptosis of glioblastoma cells by showing, for the first time, that NF-κB is critically required for Smac mimetic-mediated radiosensitization. BV6 significantly increases γ-irradiation-triggered apoptosis in several glioblastoma cell lines in a dose- and time-dependent manner. Calculation of combination index (CI) reveals that the interaction of BV6 and γ-irradiation is highly synergistic (CI < 0.3). Molecular studies show that BV6 stimulates NF-κB activation, which is critical for radiosensitization, because genetic inhibition of NF-κB by overexpression of the dominant-negative superrepressor IκBα-SR significantly decreases BV6- and γ-irradiation-induced apoptosis. Also, the BV6-mediated enhancement of γ-irradiation-triggered caspase activation, drop of mitochondrial membrane potential, and cytochrome c release is abolished in cells overexpressing IκBα-SR. Similarly, NF-κB inhibition by ectopic expression of a kinase dead mutant of IKKβ prevents the BV6-mediated sensitization for γ-irradiation. The clinical relevance is underscored by experiments with primary tumor samples showing that BV6 sensitizes primary cultured glioma cells as well as glioblastoma-initiating cancer stem cells derived from surgical specimens for γ-irradiation. In conclusion, we identify NF-κB as a critical mediator of Smac mimetic-conferred radiosensitization of glioblastoma cells. These results have important implications for the development of Smac mimetic-based combination protocols for radiosensitization of glioblastoma.     

Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.     

Kindlin-3–mediated integrin adhesion is dispensable for quiescent but essential for activated hematopoietic stem cells

Hematopoietic stem cells (HSCs) generate highly dividing hematopoietic progenitor cells (HPCs), which produce all blood cell lineages. HSCs are usually quiescent, retained by integrins in specific niches, and become activated when the pools of HPCs decrease. We report that Kindlin-3–mediated integrin activation controls homing of HSCs to the bone marrow (BM) and the retention of activated HSCs and HPCs but not of quiescent HSCs in their BM niches. Consequently, Kindlin-3–deficient HSCs enter quiescence and remain in the BM when cotransplanted with wild-type hematopoietic stem and progenitor cells (HSPCs), whereas they are hyperactivated and lost in the circulation when wild-type HSPCs are absent, leading to their exhaustion and reduced survival of recipients. The accumulation of HSPCs in the circulation of leukocyte adhesion deficiency type III patients, who lack Kindlin-3, underlines the conserved functions of Kindlin-3 in man and the importance of our findings for human disease.The entire hematopoietic system is derived from, and maintained by, a small number of hematopoietic stem cells (HSCs) that reside in the BM. HSCs are characterized by their low cycling rate and their ability to self-renew throughout the life span of an organism. After hematopoietic injury (e.g., bleeding), quiescent HSCs become activated, replenish the pool of hematopoietic effector cells, and return to the quiescent state (Trumpp et al., 2010). To maintain HSCs throughout the life of an animal, the oscillation of HSCs between quiescence, activation, self-renewal, and differentiation is precisely regulated in a specific microenvironment referred to as the stem cell niche (Morrison and Scadden, 2014). The oscillation of HSCs is regulated through interactions with niche cells (Kiel and Morrison, 2008), extracellular matrix (ECM) proteins (van der Loo et al., 1998), the action of cytokines, chemokines, and growth factors that are released by niche cells (Rizo et al., 2006), and calcium gradients established by osteoclasts during bone remodeling (Adams et al., 2006). Thus, an impairment of the HSC–niche interplay can result in loss of quiescence, uncontrolled activation, and finally exhaustion of HSCs.The interactions of HSCs with niche cells and ECM are mediated by adhesion molecules such as integrins (Wilson and Trumpp, 2006). Integrins are expressed on all cells including tissue stem cells, where they mediate binding to ECM and counter receptors (Hynes, 2002). The composition of niche cells and ECM components is unique in each organ, and hence tissue stem cells express specific integrin profiles to interact with their niche microenvironment. The integrin profile of HSCs includes multiple members of the β1 class (α2β1, α4β1, α5β1, α6β1, and α9β1), αLβ2 from the β2 class, and αvβ3 from the αv class (Grassinger et al., 2009). In vivo and in vitro studies using genetics or inhibitory antibodies demonstrated that integrins promote hematopoietic stem and progenitor cell (HSPC) homing to the BM (Potocnik et al., 2000) and their BM retention (Magnon and Frenette, 2008), proliferation, and differentiation (Arroyo et al., 1999).Integrin ligand binding and signaling require an activation step, which is induced after Talin and Kindlin bind to the cytoplasmic domains of integrin β subunits and is characterized by allosteric changes in the integrin ectodomain and transmembrane domains (Moser et al., 2009a; Shattil et al., 2010). Kindlins are evolutionarily conserved and consist of three members. Hematopoietic cells express Kindlin-3 (Ussar et al., 2006), whose deletion in mice abrogates integrin activation, resulting in hemorrhages, leukocyte adhesion defects, and osteopetrosis (Moser et al., 2008, 2009b; Schmidt et al., 2011). A human disease with similar abnormalities, called leukocyte adhesion deficiency type III (LAD-III), is also caused by null mutations of the KIND3 gene (also called FERMT3; Kuijpers et al., 2009; Malinin et al., 2009; Svensson et al., 2009).Because Kindlin-3 activates all integrins analyzed so far, it is perfectly suited to broadly abrogate integrin-dependent functions in HSCs. Here we report that proliferating hematopoietic progenitor cells (HPCs) require Kindlin-3 for their maintenance in the BM, whereas Kindlin-3–mediated integrin adhesion is dispensable for HSCs that enter quiescence. In a situation of hematopoietic stress and/or high demand for HPCs, quiescent HSCs are activated, refill the pools of HPCs, become dependent on Kindlin-3, and eventually are exhausted.     

CD160 is essential for NK-mediated IFN-γ production

NK-derived cytokines play important roles for natural killer (NK) function, but how the cytokines are regulated is poorly understood. CD160 is expressed on activated NK or T cells in humans but its function is unknown. We generated CD160-deficient mice to probe its function. Although CD160^−/− mice showed no abnormalities in lymphocyte development, the control of NK-sensitive tumors was severely compromised in CD160^−/− mice. Surprisingly, the cytotoxicity of NK cells was not impaired, but interferon-γ (IFN-γ) secretion by NK cells was markedly reduced in CD160^−/− mice. Functionally targeting CD160 signaling with a soluble CD160-Ig also impaired tumor control and IFN-γ production, suggesting an active role of CD160 signaling. Using reciprocal bone marrow transfer and cell culture, we have identified the intrinsic role of CD160 on NK cells, as well as its receptor on non-NK cells, for regulating cytokine production. To demonstrate sufficiency of the CD160⁺ NK cell subset in controlling NK-dependent tumor growth, intratumoral transfer of the CD160⁺ NK fraction led to tumor regression in CD160^−/− tumor-bearing mice, indicating demonstrable therapeutic potential for controlling early tumors. Therefore, CD160 is not only an important biomarker but also functionally controls cytokine production by NK cells.NK cells play multiple roles during the innate immune response, reacting to a myriad of challenges, including pathogen-infected cells, transplanted allogeneic cells, and tumor cells (Moretta et al., 2002; Lanier, 2005). These responses are tightly regulated through multiple activating and inhibitory receptors. Several structurally distinct receptors have been implicated in activating effector functions, including NKp46, NKG2D, 2B4 (CD244), and CS1 (CRACC; Sentman et al., 2006; Marcenaro et al., 2011). Unlike these ubiquitously expressed NK receptors, the CD160 receptor is selectively expressed on the fraction of NK cells with the highest cytotoxic functions (Maïza et al., 1993).CD160 is an immunoglobulin-like, glycosylphosphatidylinositol-anchored protein with homology to killer-cell immunoglobulin-like receptors (Agrawal et al., 1999). In addition to its association with effector function, CD160 was demonstrated to bind broadly to MHC class I molecules with low affinity, first in humans (Barakonyi et al., 2004) and later in mice (Maeda et al., 2005). A recent study, however, demonstrated that human CD160 binds to herpesvirus entry mediator (HVEM), a TNF family member, with much higher affinity than to MHC class I, and leads to suppressed T cell responses in vitro (Cai et al., 2008). Whether this high-affinity interaction exists in vivo and and what role it plays remains unclear.HVEM has been shown to regulate both the innate and adaptive responses through its multiple binding partners, both as a ligand and as a receptor. Via B and T lymphocyte attenuator (BTLA) on T cells, the delivery of HVEM is largely inhibitory, controlling T cell effector responses (Sedy et al., 2005; Deppong et al., 2006) and the innate response (Sun et al., 2009). In contrast, signaling through HVEM activates T cells by LIGHT/TNFSF14 (Cheung et al., 2005; Cai and Freeman, 2009). However, the nature of the HVEM–class I MHC–CD160 interactions has not been well defined in vivo. To directly address these questions, we generated CD160^−/− mice and soluble CD160 (CD160-Ig) fusion protein and investigated the necessity and sufficiency of CD160 on the effector function of NK cells in vivo and in vitro. We reveal here that CD160 is a functional regulator of cytokine production by NK cells and is important for early control of tumor growth.     

Lack of correlation between rejection of tumor cells co-expresssing interleukin-2 and B7.1 and vaccine efficiency

Gene modification of tumor cells to express a varietyof cytokines such as IL-2 or the co-stimulatory moleculeB7. 1 led to increased immunogenicity and reducedtumorigenicity of tumors has several models and thisprocess involves T cells. We have previously reporteddecreased tumorigenicity of the murine plasmacytomaJ558L (MHC class Ⅰ~ and class Ⅱ~-) expressing IL-2 orB7. l. When systemic immunity was analyzed,     

NK cell-mediated lysis is essential to kill Epstein–Barr virus transformed lymphoblastoid B cells when using rituximab

Rituximab is a humanized chimeric monoclonal antibody, targeted against the pan B cell marker CD20. It is frequently used to treat a variety of B cell lymphomas and immunosuppression associated lymphoproliferations such as posttransplant lymphoproliferative disorder (PTLD). The response rate of rituximab treatment is 65%, but the exact in vivo mechanism of action is not yet fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis have been suggested as effector mechanism. Rituximab may affect different types of lymphomas through different mechanisms. As lymphoblastoid cell lines (LCLs) are well-established in vitro models of PTLD, we investigated the effect of rituximab on these cells using a custom built automated laser confocal fluorescent microscope. We found that rituximab alone was not effective at inducing cell death of EBV-transformed B cells. The antibody was effective in the complement-mediated CDC. Rituximab could induce NK cell-mediated ADCC but it was more effective in the presence of untreated fresh human plasma compared to heat-inactivated human plasma. Our data suggest that complement-enhanced NK-mediated ADCC is required for effective rituximab mediated killing of EBV-transformed B cells. Determining and monitoring of serum complement levels and in vitro killing efficacy of NK cells of PTLD patients might help to predict resistant cases to rituximab therapy. On the other hand our results suggest a possibility that rituximab should be combined only with cytotoxic drugs that spare NK function when treating PTLD patients.     

Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice

Psoriasis is a common, relapsing inflammatory skin disease characterized by erythematous scaly plaques. Histological manifestations of psoriasis include keratinocyte dysregulation and hyperproliferation, elongated rete ridges, and inflammatory infiltrates consisting of T cells, macrophages, dendritic cells, and neutrophils. Despite the availability of new effective drugs to treat psoriasis, the underlying mechanisms of pathogenesis are still poorly understood. Recent studies have shown that Aldara cream, used to treat benign skin abnormalities, triggers psoriasis-like disease in humans and mice and have implicated Th17 cells in disease initiation. Using this as a model, we found a predominant role for the Th17 signature cytokines IL-17A, IL-17F, and IL-22 in psoriasiform plaque formation in mice. Using gene-targeted mice, we observed that loss of Il17a, Il17f, or Il22 strongly reduced disease the severity of psoriasis. However, we found that Th17 cells were not the primary source of these pathogenic cytokines. Rather, IL-17A, IL-17F, and IL-22 were produced by a skin-invading population of γδ T cells and RORγt(+) innate lymphocytes. Furthermore, our findings establish that RORγt(+) innate lymphocytes and γδ T cells are necessary and sufficient for psoriatic plaque formation in an experimental disease model that closely resembles human psoriatic plaque formation.     

Why is the regulation of advanced practice essential?

For many years, nurses have sought to define advanced practice (AP). There is now a developing consensus that it should be broadly defined in ways which include and embrace not only clinical practice, but also other domains such as education, management and leadership. This broad definition is congruent with Benner's notion of the novice to expert trajectory and lends support to the view that the risks around AP are minimal, since advanced practitioners by definition have expertise. The broad model of AP, however, ignores one distinct subset of advanced practice nurses who we term nurse practitioners (NPs). NPs are distinct because of their hybrid, quasi-medical practice, which crucially involves autonomous medical diagnosis and treatment. The risks in this area of practice are substantial, and consequently require nothing less than a nationally-regulated set of benchmarks. These must address the training, assessment and registration of NPs for the primary purpose of public protection.     

I mmunomodulatory effects of murine melanoma cells transduced with interleukin-2 gene and recombinant interferon-γ

The murine interleukin-2 gene was introduced intomurine melanoma B16 cells by retroviral vector. AfterG418 selection, a clone stably secreting high level ofinterleukin-2 (1038IU per 10~6 cells per 24 hours) wasobtained. The tumorigenicity of this clone decreasedsignificantly as compared with parental tumor cells andtumor cells transferred neomycin gene. There was notumor developed after injection of 5×10~5 IL2-transfectedB16 cells into C57BL/6 mice. However when the amount     

Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice

Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c⁺ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11c^hi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.The organized accumulation of lymphocytes in lymphoid organs serves to optimize both homeostatic immune surveillance and chronic responses to pathogenic stimuli (Cupedo and Mebius, 2005). During embryonic development, circulating hemopoietic cells gather at predestined sites throughout the body, where they are subsequently arranged in T and B cell–specific areas, which is characteristic of secondary lymphoid organs (SLOs). In contrast, the body seems to harbor a limited second set of selected sites that support neoformation of organized lymphoid aggregates in adult life. However, these are only revealed at times of local chronic inflammation when so-called tertiary lymphoid organs (TLOs) appear. As such, TLO was found in the pancreas of autoimmune diabetic mice (Kendall et al., 2007), around blood vessels in chronic allograft rejection (Nasr et al., 2007) and atherosclerosis (Gräbner et al., 2009), and in the brain in experimental allergic encephalitis (Magliozzi et al., 2004). In humans, TLO has been observed in the joint and lung of rheumatoid arthritis (Rangel-Moreno et al., 2006), around the airways of COPD patients (Hogg et al., 2004), and in the thyroid (Marinkovic et al., 2006). Certain infectious diseases are also accompanied by formation of TLO. Influenza virus infection of the respiratory tract leads to formation of inducible bronchus-associated lymphoid tissue (iBALT) that supports T and B cell proliferation and productive immunoglobulin class switching in germinal centers (GCs; Moyron-Quiroz et al., 2004, 2006).Although the embryonic development of SLO requires CD3⁻CD4⁺ lymphoid tissue–inducer cells, these are not a prerequisite for TLO induction (Marinkovic et al., 2006; Rangel-Moreno et al., 2007). Like SLOs, TLOs are formed in a highly regulated manner via production of homeostatic chemokines (CXCL13 and CCL19/CCL21), partially in response to signaling from the heterotrimer lymphotoxin (LT) α₁β₂ acting on the LTβ receptor on stromal lymphoid tissue organizer cells (Drayton et al., 2006). The instruction of stromal cells leads to formation of specialized high endothelial venules, and the organized production of chemokines leads to cellular organization of T cells and B cells in discrete areas. In all instances where TLOs have been described, antigen-presenting DCs have been found interspersed with T and B cell area, just as they are in SLO (Kratz et al., 1996; Cupedo et al., 2004; Moyron-Quiroz et al., 2004; Marinkovic et al., 2006; Tsuji et al., 2008). So far, the precise role of DCs in the functional organization of TLO has not been studied in great detail. Although DCs are mainly known for their function as antigen-presenting cells (Banchereau and Steinman, 1998), they are also a prominent source of homeostatic and inflammatory chemokines that can attract T and B cells and, thus, may contribute to TLO homeostasis (Beaty et al., 2007; GeurtsvanKessel and Lambrecht, 2008). In this paper, we have studied the precise contribution of DCs in the functional organization of iBALT, a specific form of TLO found in the lung after influenza virus infection (Moyron-Quiroz et al., 2004; Kocks et al., 2007).