Rong-Hua Yin

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 physically interacts with PCBP1 by anti bait coimmunoprecipitation (View interaction) THAP11 physically interacts with PCBP1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) THAP11 and PCBP1 colocalize by fluorescence microscopy (View interaction)

EDAG Positively Regulates Erythroid Differentiation and Modifies GATA1 Acetylation Through Recruiting p300

Erythroid differentiation‐associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34+ cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34+ cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO‐treated CD34+ cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG‐induced erythroid differentiation. ChIP‐PCR experiments confirmed that GATA1 and EDAG co‐occupy GATA1‐targeted genes in primary erythroid cells and in vivo. ChIP‐seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP‐Seq and GATA1 ChIP‐Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1‐activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation. Stem Cells 2014;32:2278–2289

Megakaryocytic Differentiation of K562 Cells Induced by PMA Reduced the Activity of Respiratory Chain Complex IV

Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). During the cell differentiation, the reactive oxygen species (ROS) level was increased, mitochondrial membrane potential declined and respiratory chain complex IV activity was decreased. Treatment with specific inhibitor of mitochondrial respiratory chain complex IV led to a significant inhibition in mitochondrial membrane potential and reduction of PMA-induced cell differentiation. However, treatment with cyclosporine A, a stabilization reagent of mitochondrial membrane potential, did not improve the down-regulation of mitochondrial respiratory chain complex IV induced by PMA. Furthermore, we found that the level of the complex IV core subunit COX3 and mitochondrial transport-related proteins Tim9 and Tim10 were decreased during the differentiation of K562 cells induced by PMA, suggesting an important role of these factors in mitochondrial functional changes. Our results suggest that changes in mitochondrial functions are involved in the PMA-induced K562 cell differentiation process, and the maintenance of the steady-state of mitochondrial functions plays a critical role in the regulation of cell differentiation.

GATA-2 inhibits transforming growth factor-β signaling pathway through interaction with Smad4.

GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway.

EWSR1 regulates mitosis by dynamically influencing microtubule acetylation

ABSTRACT EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

GIT2 deficiency attenuates concanavalin A‐induced hepatitis in mice

G protein‐coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein‐coupled receptors (GPCRs). The short‐splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T‐lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure inGit2 −/− mice. CD4+ T cells fromGit2 −/− mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR‐stimulated activation. Our results suggested that GIT2 plays an important role in T‐cell functionin vivo andin vitro.

Effects of THAP11 on erythroid differentiation and megakaryocytic differentiation of K562 cells.

Hematopoiesis is a complex process regulated by sets of transcription factors in a stage-specific and context-dependent manner. THAP11 is a transcription factor involved in cell growth, ES cell pluripotency, and embryogenesis. Here we showed that THAP11 was down-regulated during erythroid differentiation but up-regulated during megakaryocytic differentiation of cord blood CD34+ cells. Overexpression of THAP11 in K562 cells inhibited the erythroid differentiation induced by hemin with decreased numbers of benzidine-positive cells and decreased mRNA levels of α-globin (HBA) and glycophorin A (GPA), and knockdown of THAP11 enhanced the erythroid differentiation. Conversely, THAP11 overexpression accelerated the megakaryocytic differentiation induced by phorbol myristate acetate (PMA) with increased percentage of CD41+ cells, increased numbers of 4N cells, and elevated CD61 mRNA levels, and THAP11 knockdown attenuated the megakaryocytic differentiation. The expression levels of transcription factors such as c-Myc, c-Myb, GATA-2, and Fli1 were changed by THAP11 overexpression. In this way, our results suggested that THAP11 reversibly regulated erythroid and megakaryocytic differentiation.

Toll‐like receptor 5 signaling restrains T‐cell/natural killer T‐cell activation and protects against concanavalin A–induced hepatic injury

Toll‐like receptor‐5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)–mediated liver injury. Here, we show that TLR5 is highly up‐regulated in the hepatic mononuclear cells of mice during Con A–induced hepatitis. Increased mortality and liver histopathology of TLR5‐deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A–induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A–mediated hepatitis in wild‐type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T‐cell/natural killer T‐cell activity and cytokine production in the Con A–hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow–derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A–induced liver injury. Moreover, interleukin‐6 elevation induced by CBLB502 is an important protective factor against Con A–induced liver injury. In addition, we demonstrate that CBLB502 suppresses α‐galactosylceramide‐induced natural killer T cell–dependent inflammatory liver injury. Conclusion: The TLR5 signaling pathway plays an important role in T cell–mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059‐2073).

TLR5 signaling restrains T/NKT cell activation and protects against concanavalin A‐induced hepatic injury

Toll‐like receptor‐5 (TLR5) signaling regulates the immune privileged status of the liver and is involved in hepatic immune disorders. However, the role of TLR5 has not yet been investigated in experimental models of concanavalin A (Con A)–mediated liver injury. Here, we show that TLR5 is highly up‐regulated in the hepatic mononuclear cells of mice during Con A–induced hepatitis. Increased mortality and liver histopathology of TLR5‐deficient mice correlated with excessive production of proinflammatory cytokines, suggesting that TLR5 knockout mice were more susceptible to Con A–induced hepatitis. We also report that administration of CBLB502, an exogenous TLR5 agonist, substantially alleviated Con A–mediated hepatitis in wild‐type mice as shown by increased survival rates, reduced aminotransferase and proinflammatory cytokine production, impaired lymphocyte infiltration, and ameliorated hepatocyte necrosis and/or apoptosis. Mechanistic studies revealed that CBLB502 acts as a negative regulator in limiting T‐cell/natural killer T‐cell activity and cytokine production in the Con A–hepatitis model. Bone marrow transplantation experiments showed that TLR5 in bone marrow–derived cells contributed to the hepatoprotective efficacy of CBLB502 against Con A–induced liver injury. Moreover, interleukin‐6 elevation induced by CBLB502 is an important protective factor against Con A–induced liver injury. In addition, we demonstrate that CBLB502 suppresses α‐galactosylceramide‐induced natural killer T cell–dependent inflammatory liver injury. Conclusion: The TLR5 signaling pathway plays an important role in T cell–mediated hepatic injury and may be exploited for therapeutic treatment of inflammatory liver diseases. (Hepatology 2017;65:2059‐2073).

Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

BACKGROUND & AIMS Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. METHODS AND RESULTS During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. CONCLUSIONS Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis.