Crystal Structure of the Ubiquitin-like Domain-CUT Repeat-like Tandem of Special AT-rich Sequence Binding Protein 1 (SATB1) Reveals a Coordinating DNA-binding Mechanism

SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1–248, SATB1^(1–248)), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.     

Fas-Mediated Apoptosis Is Regulated by the Extracellular Matrix Protein CCN1 (CYR61) In Vitro and In Vivo

Although Fas ligand (FasL) is primarily expressed by lymphoid cells, its receptor Fas (CD95/Apo-1) is broadly expressed in numerous nonlymphoid tissues and can mediate apoptosis of parenchymal cells upon injury and infiltration of inflammatory cells. Here we show that CCN1 (CYR61) and CCN2 (CTGF), matricellular proteins upregulated at sites of inflammation and wound repair, synergize with FasL to induce apoptosis by elevating cellular levels of reactive oxygen species (ROS). CCN1 acts through engagement of integrin α₆β₁ and cell surface heparan sulfate proteoglycans, leading to ROS-dependent hyperactivation of p38 mitogen-activated protein kinase in the presence of FasL to enhance mitochondrial cytochrome c release. We show that CCN1 activates neutral sphingomyelinase, which functions as a key source of CCN1-induced ROS critical for synergism with FasL. Furthermore, Fas-dependent hepatic apoptosis induced by an agonistic monoclonal anti-Fas antibody or intragastric administration of alcohol is severely blunted in knock-in mice expressing an apoptosis-defective Ccn1 allele. These results demonstrate that CCN1 is a physiologic regulator of Fas-mediated apoptosis and that the extracellular matrix microenvironment can modulate Fas-dependent apoptosis through CCN1 expression.Cell adhesion to several abundant extracellular matrix (ECM) proteins via engagement of integrin receptors is known to induce potent prosurvival signals, whereas detachment from the ECM triggers many cell types to undergo anoikis, a form of apoptotic cell death (13). This regulation of cell survival through integrin-mediated cell adhesion plays a critical role in controlling homeostasis and the integrity of tissue architecture, whereas unligated or inappropriately ligated integrins may elicit apoptotic signals (12). However, during embryogenesis, inflammation, tissue remodeling, and wound repair, death-inducing factors can provoke programmed or apoptotic death in normal cells without requiring their detachment from the ECM (4).Fas (CD95/APO-1) is a member of the tumor necrosis factor (TNF) receptor family of cell surface death receptors that mediates apoptotic signals upon binding to its specific ligand, FasL. Ligation of Fas to FasL or its agonistic antibodies results in receptor clustering, recruitment of the adaptor protein FADD, and activation of the proteolytic caspase cascade (19, 50). Whereas FasL is primarily expressed in activated T lymphocytes, natural killer cells, and tissues of immune privilege, Fas is broadly expressed in most lymphoid and nonlymphoid tissues (50). Fas-mediated apoptosis is critical for the regulation of the immune response, including deletion of activated T and B lymphocytes, cell death-inducing activity of cytotoxic T cells, and removal of infiltrating lymphocytes in immune-privileged tissues (19, 50). Fas also plays an important role in parenchymal cell apoptosis in many organs during tissue injury and upon inflammatory infiltration of lymphocytes (7, 20, 38, 42, 46). Consistent with the notion that cell adhesion promotes cell survival, integrin-matrix interactions inhibit Fas-dependent apoptosis in a variety of cell types (22, 32). Thus, optimal apoptotic responses to Fas/FasL signaling in adherent parenchymal cells must override the cytoprotective effects of integrin-mediated cell adhesion. In these instances, dynamic changes in the ECM induced by inflammation or injury repair may establish conditions that are permissive of, or conducive to, the apoptotic responses to FasL.Recent studies have described the emergence of ECM proteins that can induce or promote apoptosis (49, 60, 65). Among them are members of the CCN family (9), which are secreted cysteine-rich proteins that serve regulatory rather than structural roles in the ECM and are therefore considered matricellular proteins (6). CCN1 (CYR61) and CCN2 (CTGF) support cell adhesion, stimulate cell migration, induce angiogenesis, and promote chondrogenic differentiation, exerting their functions primarily through direct binding to integrin receptors. CCN1 and CCN2 promote the survival of endothelial cells through integrin α_vβ₃ but induce apoptosis in p21-deficient fibroblasts through α₆β₁ via a caspase-8-independent mechanism (3, 40, 60). CCN1 and CCN2 are also critical for embryonic development, as Ccn1-null mice die during midgestation due to cardiovascular abnormalities and Ccn2-deficient mice perish perinatally as a consequence of severe skeletal malformations (29, 47, 48). In the adult, CCN proteins are highly expressed at sites of inflammation, injury repair, and tissue remodeling and are implicated in diseases where inflammation plays a role, including fibrosis, atherosclerosis, arthritis, and cancer (9). Furthermore, the presence of CCN1 in the ECM enables TNF-α to induce apoptotic death in normal cells without inhibition of NF-κB signaling or de novo protein synthesis, conditions thought to be necessary for TNF-α to be cytotoxic (10).Here we show that CCN1 and CCN2 can synergize with FasL and significantly enhance FasL-induced apoptosis in fibroblasts. Mechanistically, CCN1 engages integrin α₆β₁ and cell surface heparan sulfate proteoglycans (HSPGs), leading to the reactive oxygen species (ROS)-dependent hyperactivation of p38 mitogen-activated protein kinase (MAPK) in the presence of FasL, which greatly enhances mitochondrial cytochrome c release and apoptosis. We show that CCN1 is a novel activator of neutral sphingomyelinase (nSMase), which is an essential contributor to CCN1-induced ROS. Further, Fas-dependent hepatic cell death is greatly diminished in knock-in mice expressing an apoptosis-defective mutant of CCN1 that is unable to bind α₆β₁-HSPGs. Together, these results show that CCN1 is a physiologic regulator of Fas-mediated apoptosis and indicate that Fas-dependent cell death at sites of inflammation and injury repair may be controlled by the matrix microenvironment through CCN1 expression.     

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.     

The HIV-1 Tat Protein Induces the Activation of CD8+ T Cells and Affects In Vivo the Magnitude and Kinetics of Antiviral Responses

T cells are functionally compromised during HIV infection despite their increased activation and proliferation. Although T cell hyperactivation is one of the best predictive markers for disease progression, its causes are poorly understood. Anti-tat natural immunity as well as anti-tat antibodies induced by Tat immunization protect from progression to AIDS and reverse signs of immune activation in HIV-infected patients suggesting a role of Tat in T cell dysfunctionality. The Tat protein of HIV-1 is known to induce, in vitro, the activation of CD4⁺ T lymphocytes, but its role on CD8⁺ T cells and how these effects modulate, in vivo, the immune response to pathogens are not known. To characterize the role of Tat in T cell hyperactivation and dysfunction, we examined the effect of Tat on CD8⁺ T cell responses and antiviral immunity in different ex vivo and in vivo models of antigenic stimulation, including HSV infection. We demonstrate for the first time that the presence of Tat during priming of CD8⁺ T cells favors the activation of antigen-specific CTLs. Effector CD8⁺ T cells generated in the presence of Tat undergo an enhanced and prolonged expansion that turns to a partial dysfunctionality at the peak of the response, and worsens HSV acute infection. Moreover, Tat favors the development of effector memory CD8⁺ T cells and a transient loss of B cells, two hallmarks of the chronic immune activation observed in HIV-infected patients. Our data provide evidence that Tat affects CD8⁺ T cell responses to co-pathogens and suggest that Tat may contribute to the CD8⁺ T cell hyperactivation observed in HIV-infected individuals.     

In Vitro and In Vivo Enhancement of Adipogenesis by Italian Ryegrass (Lolium multiflorum) in 3T3-L1 Cells and Mice

Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as α-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-γ2, CCAAT/enhancer binding protein-α, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPARγ-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals.     

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.     

Nature of Escherichia coli B(P1) Yielder Cells at the Time of Infection with Restricted T1

In the infection of Escherichia coli B(P1) with restricted T1, it was shown that yielder cells consist of both special and nonspecial cells. Special or predetermined yielders occurred only among the earliest yielders. In most instances, yielder-cell formation was most easily explained by assuming that the first step was a chance escape of the restricted phage DNA from the degrading enzyme of the restricting cell.     

Initiation of Antiretroviral Therapy (ART) at Different Stages of HIV-1 Disease Is Not Associated with the Proportion of Exhausted CD8+ T Cells

CD8⁺ T cell-restricted immunity is important in the control of HIV-1 infection, but continued immune activation results in CD8⁺ T cell dysfunction. Early initiation of antiretroviral treatment (ART) and the duration of ART have been associated with immune reconstitution. Here, we evaluated whether restoration of CD8⁺ T cell function in HIV-1-infected individuals was dependent on early initiation of ART. HIV-specific CD107a, IFNγ, IL-2, TNFα and MIP-1β expression by CD8⁺ T cells and the frequency of CD8⁺ T cells expressing PD-1, 2B4 and CD160 were measured by flow cytometry. The frequency of CD8⁺ T cells expressing the inhibitory markers PD-1, 2B4 and CD160 was lower in ART-treated individuals compared with ART-naïve individuals and similar to the frequency in HIV-uninfected controls. The expression of the three markers was similarly independent of when therapy was initiated. Individuals treated before seroconversion displayed an HIV-specific CD8⁺ T cell response that included all five functional markers; this was not observed in individuals treated after seroconversion or in ART-naïve individuals. In summary, ART appears to restore the total CD8⁺ T cell population to a less exhausted phenotype, independent of the time point of initiation. However, to preserve multifunctional, HIV-1-specific CD8⁺ T cells, ART might have to be initiated before seroconversion.     

Genome-wide Approaches Reveal Functional Vascular Endothelial Growth Factor (VEGF)-inducible Nuclear Factor of Activated T Cells (NFAT) c1 Binding to Angiogenesis-related Genes in the Endothelium

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.     

Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1-Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), ‘RPRK’, within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, G_αi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.     

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).     

Inhibition of Protein Phosphatase 2A (PP2A) Prevents Mcl-1 Protein Dephosphorylation at the Thr-163/Ser-159 Phosphodegron,Dramatically Reducing Expression in Mcl-1-amplified Lymphoma Cells

Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.     

T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.