Special AT-rich sequence-binding protein 2 acts as a negative regulator of stemness in colorectal cancer cells

AIMTo find the mechanisms by which special AT-rich sequence-binding protein 2 (SATB2) influences colorectal cancer (CRC) metastasis.METHODSCell growth assay, colony-forming assay, cell adhesion assay and cell migration assay were used to evaluate the biological characteristics of CRC cells with gain or loss of SATB2. Sphere formation assay was used to detect the self-renewal ability of CRC cells. The mRNA expression of stem cell markers in CRC cells with upregulated or downregulated SATB2 expression was detected by quantitative real-time polymerase chain reaction. Chromatin immunoprecipitation (ChIP) was used to verify the binding loci of SATB2 on genomic sequences of stem cell markers. The Cancer Genome Atlas (TCGA) database and our clinical samples were analyzed to find the correlation between SATB2 and some key stem cell markers.RESULTSDownregulation of SATB2 led to an aggressive phenotype in SW480 and DLD-1 cells, which was characterized by increased migration and invasion abilities. Overexpression of SATB2 suppressed the migration and invasion abilities in SW480 and SW620 cells. Using sequential sphere formation assay to detect the self-renewal abilities of CRC cells, we found more secondary sphere formation but not primary sphere formation in SW480 and DLD-1 cells after SATB2 expression was knocked down. Moreover, most markers for stem cells such as CD133, CD44, AXIN2, MEIS2 and NANOG were increased in cells with SATB2 knockdown and decreased in cells with SATB2 overexpression. ChIP assay showed that SATB2 bound to regulatory elements of CD133, CD44, MEIS2 and AXIN2 genes. Using TCGA database and our clinical samples, we found that SATB2 was correlated with some key stem cell markers including CD44 and CD24 in clinical tissues of CRC patients.CONCLUSIONSATB2 can directly bind to the regulatory elements in the genetic loci of several stem cell markers and consequently inhibit the progression of CRC by negatively regulating stemness of CRC cells.     

Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.

Zika virus (ZIKV), a mosquito-borne enveloped RNA virus that belongs to the Flaviviridae family, has gained notoriety recently, due to its explosive outbreaks and association with serious clinical diseases, such as Guillain-Barré syndrome in adults and microcephaly in newborns (1–4). Currently, no ZIKV-specific therapies or prophylactic vaccines are available (5). ZIKV genome is a positive-sense, single-stranded RNA [ssRNA(+)] (6). The viral replication occurs on the surface of the endoplasmic reticulum (ER), where the double-strand RNA (dsRNA) is synthesized from viral genomic ssRNA(+) and transcribed into new proteins (7, 8). The viral genome is translated into a polyprotein, which is proteolytically processed into three structural proteins (capsid [C], precursor membrane [prM], and envelop [Env]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), by both host and viral proteases (9, 10). ZIKV infection can rewire cellular components to establish viral replication complexes (RCs), which increase the local concentration of viral and cellular factors for efficient viral replication (11–13). However, the function of rewired cytoskeleton network remains elusive in ZIKV infection.Vimentin is the most abundant intermediate filaments (IFs), which generally surrounds the nucleus and extends throughout the cytoplasm, providing help to important biological processes, such as organelle positioning, cell migration, and cell signaling (14). Except for acting as an integrator of cellular mechanical functions, the highly dynamic nature of vimentin filaments enables it to respond rapidly to pathological stimuli (15). Several studies have observed the phenomenon of vimentin network rearrangement in viral infections and proposed that the role of intact vimentin scaffold could contribute to the viral life cycle (16–21). However, evidence for the dynamic changes of vimentin IFs during ZIKV infection and its contribution to RCs integrity and stability remain understudied.In addition to providing a structural scaffold, there is evidence indicating that cytoskeletal proteins also regulate translational apparatus (22). For example, ribosomes can physically associate with microtubules (MTs) and F-actin in different cells (23, 24). Disorganization of F-actin by cytochalasin D impairs local protein synthesis in isolated axoplasmic nerve fibers (25). The interaction between keratin IFs and the Y subunit of eukaryotic elongation factor-1 (eEF1BY) plays an essential role in protein synthesis (26). MTs can bind to the cytoplasmic tail of RRBP1 and take part in ER organization and neuronal polarity (27, 28). However, information on the spatial and functional relationship between vimentin IFs and the translational machinery, especially in the context of virus infection, remain completely unknown.In this study, we investigated the function of vimentin IFs in ZIKV infection. By monitoring spatial-temporal responses of the cellular vimentin network throughout various steps of the ZIKV life cycle, we demonstrated that ZIKV infection induces massive rearrangements of cytoplasmic vimentin. When vimentin protein was genetically depleted from cells, distribution of viral proteins is scattered within infected cells, indicating its “organizer” role. Viral RNA replication, protein synthesis, and virion release are subsequently reduced. Using mass spectrometry (MS) and RNA-sequencing (RNA-seq) analysis, we discovered many intimate interactions between vimentin and RNA-binding proteins (RBPs), and that vimentin facilitates ZIKV RNA replication by interacting with and regulating the level and subcellular distribution of RRBP1, indicating its “regulator” role. Thus, our work establishes important connections among vimentin filaments dynamics, ZIKV RCs, and cellular RBPs in highly effective infection.     

Beta(2)-microglobulin as a negative growth regulator of myeloma cells

High beta(2)-microglobulin (beta(2)m) levels in myeloma correlate with poor prognosis. We hypothesized that beta(2)m may affect myeloma cell growth and survival. In this study, we examined the in vitro effects of beta(2)m on myeloma cells. Primary myeloma cells freshly isolated from patients and myeloma cell lines were used, cultured in the presence of beta(2)m, and monitored for growth and survival. Beta(2)m suppressed the growth of primary tumour cells and myeloma cell lines (ARK-RS, ARP-1, RPMI-8226, U266, ARH-77 and IM-9). High concentrations of beta(2)m induced apoptosis and cell cycle arrest. Beta(2)m-induced apoptosis was dependent on activation of a caspase cascade, inhibited by interleukin 6, and did not involve the surface death receptors, as receptor-neutralizing antibodies had no inhibitory effect. Beta(2)m-induced growth arrest was associated with downregulation of cyclins A and D2. Surprisingly, anti-beta(2)m antibodies did not block the effect of beta(2)m but were synergistic with beta(2)m, resulting in 90% growth inhibition and 70% apoptosis of myeloma cells. Whereas beta(2)m treatment resulted in slight upregulation of surface beta(2)m and major histocompatibility complex class I alpha-chain expression, treatment of myeloma cells with anti-beta(2)m antibodies alone or with beta(2)m resulted in significant downregulation of surface beta(2)m and class I molecules, suggesting that class I molecules may be involved in signal transduction. Our data demonstrate that beta(2)m plays an important role in regulating the growth and survival of myeloma cells in vitro and warrants further investigation to delineate the mechanisms of beta(2)m and anti-beta(2)m antibody-induced growth regulation of myeloma cells.     

Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.

Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.     

The proapoptotic and antimitogenic protein p66SHC acts as a negative regulator of lymphocyte activation and autoimmunity

The ShcA locus encodes 3 protein isoforms that differ in tissue specificity, subcellular localization, and function. Among these, p66Shc inhibits TCR coupling to the Ras/MAPK pathway and primes T cells to undergo apoptotic death. We have investigated the outcome of p66Shc deficiency on lymphocyte development and homeostasis. We show that p66Shc^–/– mice develop an age-related lupus-like autoimmune disease characterized by spontaneous peripheral T- and B-cell activation and proliferation, autoantibody production, and immune complex deposition in kidney and skin, resulting in autoimmune glomerulonephritis and alopecia. p66Shc^–/– lymphocytes display enhanced proliferation in response to antigen receptor engagement in vitro and more robust immune responses both to vaccination and to allergen sensitization in vivo. The data identify p66Shc as a negative regulator of lymphocyte activation and show that loss of this protein results in breaking of immunologic tolerance and development of systemic autoimmunity.      

Borrelia burgdorferi--induced oxidative burst, calcium mobilization, and phagocytosis of human neutrophils are complement dependent

When Borrelia burgdorferi, the spirochete causing Lyme disease, is transmitted to a human, the complement system is among the first challenges facing the bacterium. Neutrophils are crucial leukocytes in the first line of host defense against bacterial infections. To investigate the role of complement in the Borrelia-induced activation of human neutrophils, oxidative burst, calcium mobilization, and phagocytosis induced by three subspecies of B. burgdorferi were studied. Each subspecies induced all observed neutrophil functions in a complement-dependent manner. Serum-derived factors bound to the surface of B. burgdorferi were found to be essential for the induction of the oxidative burst. The CD11b chain of CR3 was found to participate in the oxidative burst and calcium mobilization induced by B. burgdorferi.     

Protein phosphatase 5 as a negative key regulator of Raf-1 activation

Growth factors such as the epidermal growth factor cause sequential activation of receptor tyrosine kinases, adaptor molecules and the Raf-MEK-ERK pathway. The kinetics and intensity of these signals are dependent on the balance between phosphorylation and dephosphorylation of these molecules by numerous kinases and phosphatases, respectively. Recently, protein phosphatase 5 has been characterized as a key dephosphorylation regulator of Raf-1 activation in growth factor-mediated signaling, leading to attenuation of the MEK-ERK cascade.     

The A-kinase anchor protein AKAP121 is a negative regulator of cardiomyocyte hypertrophy

Pathologic cardiac hypertrophy imposes a significant clinical burden on patients, yet the precise intracellular mechanisms responsible for its induction are only partially understood. We examined a potential role for AKAP121 to regulate cardiomyocyte hypertrophy, since recent reports have implicated other AKAPs in this process. We report here that knockdown of AKAP121 expression in isolated neonatal rat cardiomyocytes results in pronounced cellular hypertrophy. Loss of AKAP121 expression is associated with dephosphorylation and nuclear localization of NFATc3, a downstream effector of the hypertrophic phosphatase calcineurin. We also demonstrate that over-expression of AKAP121 in cardiac myocytes reduces basal cell size, and blocks hypertrophy induced by isoproterenol, indicating that AKAP121 negatively regulates the hypertrophic process. Co-immunoprecipitation data indicates that AKAP121 and calcineurin directly interact. Our findings are consistent with a model in which loss of AKAP121 expression leads to the release of an active pool of calcineurin, in turn causing nuclear translocation of NFATc3 and activation of the hypertrophic gene program. These results are the first to identify AKAP121 as a negative regulator of cardiomyocyte hypertrophy, and highlight AKAP121 as a potential target for therapeutic exploitation.