The TGFß→TAK1→LATS→YAP1 Pathway Regulates the Spatiotemporal Dynamics of YAP1.

The Hippo kinase cascade functions as a central hub that relays input from the "outside world" of the cell and translates it into specific cellular responses by regulating the activity of Yes-associated protein 1 (YAP1). How Hippo translates input from the extracellular signals into specific intracellular responses remains unclear. Here, we show that transforming growth factor ß (TGFß)-activated TAK1 activates LATS1/2, which then phosphorylates YAP1. Phosphorylated YAP1 (p-YAP1) associates with RUNX3, but not with TEAD4, to form a TGFß-stimulated restriction (R)-point-associated complex which activates target chromatin loci in the nucleus. Soon after, p-YAP1 is exported to the cytoplasm. Attenuation of TGFß signaling results in re-localization of unphosphorylated YAP1 to the nucleus, where it forms a YAP1/TEAD4/SMAD3/AP1/p300 complex. The TGFß-stimulated spatiotemporal dynamics of YAP1 are abrogated in many cancer cells. These results identify a new pathway that integrates TGFß signals and the Hippo pathway (TGFß→TAK1→LATS1/2→YAP1 cascade) with a novel dynamic nuclear role for p-YAP1.

Bisphenol A-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression by Syk activation.

The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving immune system alterations, but the underlying mechanisms have yet to be elucidated. We previously reported that BPA at concentration of 100 µM caused human B cell death in accordance with an increase in nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression. Autophagy is a cellular process that degraded and recycles cytoplasmic constituents. Here, we investigated whether BPA induces autophagy through Nrf2, which is associated with regulation of B cell death using human WiL2-NS lymphoblast B cells. Then, cell viability was assessed by various assays using trypan blue, MTT or Celltiter glo luminescent substrate and DAPI. When WiL2-NS cells were treated with BPA, cell viability was decreased and LC3 autophagy cargo protein/puncta was increased. BPA-induced autophagy was confirmed by the modification of LC3 puncta formation or autophagy flux turnover with the treatment of hydroxychloroquine(HCQ), NH4Cl and PI3K inhibitors including 3-methyladenine(3-MA), LY294002 and wortmannin. BPA treatment increased the expression of autophagy-related gene(Atg)7 and Beclin1 as well as Nrf2 induced by the production of reactive oxygen species (ROS). The inhibition of autophagy with siAtg7 or siBeclin1 and Nrf2 depletion aggravated BPA-induced cell death. BPA enhanced the bound of Nrf2 to the specific region on Beclin1 and Atg7 promoter. Spleen tyrosine kinase(Syk) activity was enhanced in response to BPA treatment. Bay61-3606, Syk inhibitor, decreased LC3 and the expression of Atg7 and Beclin1, leading to the increase of BPA-induced B cell death. The results suggest that BPA-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression.

Spleen tyrosine kinase regulates crosstalk of hypoxia-inducible factor-1α and nuclear factor (erythroid-derived2)-like 2 for B cell survival.

B cells play a major role in regulating disease incidence through various factors, including spleen tyrosine kinase (Syk), which transmits signals to all hematopoietic lineage cells. Hypoxia-inducible factor (HIF)-1α accumulates under hypoxic conditions, which is also oxidative stress to induce nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responsible for gene expression of antioxidant enzymes. In the present study, we investigated whether B cells are regulated by crosstalk of HIF-1α and Nrf2 via reactive oxygen species (ROS)-mediated Syk activation. When B cells were incubated under hypoxic conditions, Syk phosphorylation, HIF-1α, and Nrf2 levels were increased. Hypoxia-inducible results were consistent with CoCl2 treatment, which mimics hypoxic conditions. Cell viability was reduced by the Syk inhibitor BAY 61-3606. Increased Nrf2 levels due to hypoxia or CoCl2 were inhibited by treatment with a HIF inhibitor. Hypoxia- or CoCl2-induced ROS increased HIF-1α and Nrf2 levels, which were attenuated by treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. HIF-1α levels were reduced in doxycycline-treated shNrf2 cells. Clobetasol propionate, a Nrf2 inhibitor, also inhibited HIF-1α levels induced by hypoxia or CoCl2. ROS-mediated Syk phosphorylation at tyrosine 525/526 was confirmed by treatment with H2O2, hypoxia, and CoCl2, and attenuated with NAC treatment. Inhibition of Syk phosphorylation by BAY 61-3606 is consistent with a decrease in protein HIF-1α and Nrf2 levels. Taken together, HIF-1α levels might control Nrf2 levels and vice versa, and could be associated with Syk phosphorylation in B cells. The results indicate that B cells could be regulated by crosstalk of HIF-1α and Nrf2 through ROS-mediated Syk activation.

CXCR4 promotes B cell viability by the cooperation of nuclear factor (erythroid-derived 2)-like 2 and hypoxia-inducible factor-1α under hypoxic conditions.

B cells that interact with T cells play a role in regulating the defense function by producing antibodies and inflammatory cytokines. C-X-C chemokine receptor type 4 (CXCR4) is a specific receptor for stromal cell-derived factor 1 (SDF-1) that controls various B cell functions. Here, we investigated whether CXCR4 regulates B cell viability by inducing hypoxia-inducible factor (HIF)-1α and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) under a hypoxic condition in WiL2-NS human B cells. Nrf2 and CXCR4 expressions increased significantly when WiL2-NS cells were incubated under a hypoxic condition. Interfering with CXCR4 expression using CXCR4-siRNA inhibited cell viability. CXCR4 expression also decreased after treatment with a HIF inhibitor under the hypoxic condition, leading to inhibited cell viability. Increased reactive oxygen species (ROS) levels and the expression of HIF-1α and Nrf2 decreased under the hypoxic condition following incubation with N-acetylcysteine, a ROS scavenger, which was associated with a decrease in CXCR4 expression. CXCR4 expression was augmented by overexpressing Nrf2 after transfecting the pcDNA3.1-Nrf2 plasmid. CXCR4 expression decreased and HIF-1α accumulation decreased when Nrf2 was inhibited by doxycycline in tet-shNrf2-expressed stable cells. Nrf2 or HIF-1α bound from -718 to -561 of the CXCR4 gene promoter as judged by a chromatin immunoprecipitation assay. Taken together, these data show that B cell viability under a hypoxic condition could be regulated by CXCR4 expression through binding of HIF-1α and Nrf2 to the CXCR4 gene promoter cooperatively. These results suggest that CXCR4 could be an additional therapeutic target to control B cells with roles at disease sites under hypoxic conditions.

RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis.

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFß and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.

BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts.

Cell survival is facilitated by the maintenance of mitochondrial membrane potential (MMP). B cell activating factor (BAFF) plays a role in survival, differentiation, and maturation of B cells. In the present study, we examined whether BAFF could attenuate oxidative stress-induced B cell death by the regulation of MMP collapse via spleen tyrosine kinase (Syk) activation using WiL2-NS human B lymphoblast cells. BAFF binds to receptors on WiL2-NS cells. When the cells were incubated in serum-deprived conditions with 1% fetal bovine serum (FBS), BAFF reduced the percentage of dead cells as determined through trypan blue staining and caspase 3 activity. BAFF also inhibited MMP collapse with 1% FBS, as indicated by a decrease in the number of cells with high-red fluorescence of MitoProbe™ JC-1 reagent or a decrease in the percentage of DiOC6-stained cells. Reactive oxygen species (ROS) production was reduced by incubation with BAFF in the presence of 10% or 1% FBS. BAFF inhibited MMP collapse, cell growth retardation, dead cell formation, and caspase 3 activation caused by treatment with H2O2. Syk phosphorylation on tyrosine (Y) 525/526 was increased in cells incubated with 1% FBS in the presence of BAFF than cells incubated with 1% FBS or BAFF alone. BAY61-3606, a Syk inhibitor reduced the effect of BAFF on MMP collapse, caspase 3 activation, cell growth retardation, and dead cell formation. Together, these data demonstrate that BAFF might attenuate oxidative stress-induced B cell death and growth retardation by the maintenance of MMP through Syk activation by Y525/526 phosphorylation. Therefore, BAFF and Syk might be therapeutic targets in the pathogenesis of B cell-associated diseases such as autoimmune disease.

Bisphenol A and its substitutes regulate human B cell survival via Nrf2 expression.

B cells contribute to produce inflammatory cytokines and antibodies, to present autoantigens, and to interact with T cells, which lead to body defense and disease control. Nuclear factor (erythroid-derived 2)-like 2(Nrf2) is responsible for gene expression of antioxidant enzymes to protect cells from oxidative stress by reactive oxygen species(ROS) production. Bisphenol A(BPA) may not be safe due to the effect on body's physiological functions. The chemicals that substitute for BPA may still have similar effects in the body. Tritan™ copolyester is a novel plastic form using BPA substitutes, 1,4-cyclohexanedimethanol(CHDM), dimethyl terephthalate(DMT), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol(TMCD). Isosorbide(ISO) was also used as a substitute for TMCD and DMT. Here, we investigated whether B cell viability is influenced by BPA and its substitutes via Nrf2 induction using WiL2-NS human B lymphoblast cells. When cytotoxicity was measured by using assays with MTT, CellTiter-Glo, trypan blue and propidium iodide, cytotoxicity by BPA was higher than that by substitutes. BPA and its substitutes showed significant cytotoxicity and ROS production, which were attenuated by the treatment with N-acetylcysteine(NAC), a ROS scavenger. In addition, BPA treatment enhanced gene expression of antioxidant enzymes, heme oxygenase(HO)-1, catalase, superoxide dismutase(SOD) 1 and 2. As H2O2 treatment induced cell death and Nrf2 amount in WiL2-NS cells, BPA treatment increased Nrf2. Cell death by H2O2 was increased in doxycycline-inducible Nrf2-knockdown(KD) cells. In Cytotoxicity by the treatment with BPA or its substitutes was also enhanced in Nrf2-KD cells but that was reduced by Nrf2 overexpression compared to control cells. Taken together, these results implicate that B cell cytotoxicity by substitutes should be lower than BPA and Nrf2 can prevent B cells from BPA- or BPA substitutes-induced cytotoxicity via ROS production. Data suggest that the comprehensive studies or evaluation could be necessary to replace BPA in manufacture by other substitutes.

Reciprocal regulation of YAP/TAZ by the Hippo pathway and the Small GTPase pathway.

Yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) (YAP/TAZ) are transcriptional coactivators that regulate genes involved in proliferation and transformation by interacting with DNA-binding transcription factors. Remarkably, YAP/TAZ are essential for cancer initiation or growth of most solid tumors. Their activation induces cancer stem cell attributes, proliferation, and metastasis. The oncogenic activity of YAP/TAZ is inhibited by the Hippo cascade, an evolutionarily conserved pathway that is governed by two kinases, mammalian Ste20-like kinases 1/2 (MST1/2) and Large tumor suppressor kinase 1/2 (LATS1/2), corresponding to Drosophila's Hippo (Hpo) and Warts (Wts), respectively. One of the most influential aspects of YAP/TAZ biology is that these factors are transducers of cell structural features, including polarity, shape, and cytoskeletal organization. In turn, these features are intimately related to the cell's ability to attach to other cells and to the surrounding extracellular matrix (ECM), and are also influenced by the cell's microenvironment. Thus, YAP/TAZ respond to changes that occur at the level of whole tissues. Notably, small GTPases act as master organizers of the actin cytoskeleton. Recent studies provided convincing genetic evidence that small GTPase signaling pathways activate YAP/TAZ, while the Hippo pathway inhibits them. Biochemical studies showed that small GTPases facilitate the YAP-Tea domain transcription factor (TEAD) interaction by inhibiting YAP phosphorylation in response to serum stimulation, while the Hippo pathway facilitates the YAP-RUNX3 interaction by increasing YAP phosphorylation. Therefore, small GTPase pathways activate YAP/TAZ by switching its DNA-binding transcription factors. In this review, we summarize the relationship between the Hippo pathway and small GTPase pathways in the regulation of YAP/TAZ.

RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point.

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

Spleen tyrosine kinase-dependent Nrf2 activation regulates oxidative stress-induced cell death in WiL2-NS human B lymphoblasts.

Autoimmune rheumatic lesions are often characterised by the immune cell recruitment including B lymphocytes and the presence of reactive oxygen species (ROS), which increase antioxidant gene transcription via nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Spleen tyrosine kinase (Syk) has a major role in the signal transmission of all haematopoietic lineage cells including B/T cells, mast cells, and macrophages. In this study, we investigated whether B cell survival is regulated by Nrf2 via ROS-mediated Syk activation in WiL2-NS human B lymphoblast cells. When WiL2-NS cells were incubated with 1% foetal bovine serum (FBS), the survival rate and mitochondrial membrane potential (MMP) were reduced. In addition, 1% FBS increased caspase 3 activity, cytochrome C release, nuclear localisation of Nrf2, and ROS production. N-acetylcysteine attenuated ROS production and nuclear translocation of Nrf2. It also inhibited cell death, caspase 3 activation, MMP collapse, and cytochrome C release. Results from the 1% FBS treatment were consistent with those of H2O2 treatment. Syk phosphorylation at tyrosine 525/526 was increased by incubation with 1% FBS or treatment with 100 µM H2O2. Nuclear translocation of Nrf2 by H2O2 was inhibited by treatment with BAY61-3606, a Syk inhibitor. BAY61-3606 also promoted MMP collapse, cytochrome C release, caspase 3 activation, and cell death. Taken together, these results implicate that Syk controls oxidative stress-induced human B cell death via nuclear translocation of Nrf2 and MMP collapse. These results suggest that Syk is a novel regulator of Nrf2 activation.

DNA binding partners of YAP/TAZ.

Hippo signaling plays critical roles in regulation of tissue homeostasis, organ size, and tumorigenesis by inhibiting YES-associated protein (YAP) and PDZ-binding protein TAZ through MST1/2 and LATS1/2 pathway. It is also engaged in cross-talk with various other signaling pathways, including WNT, BMPs, Notch, GPCRs, and Hedgehog to further modulate activities of YAP/TAZ. Because YAP and TAZ are transcriptional coactivators that lack DNA-binding activity, both proteins must interact with DNA-binding transcription factors to regulate target gene's expression. To activate target genes involved in cell proliferation, TEAD family members are major DNA-binding partners of YAP/TAZ. Accordingly, YAP/TAZ were originally classified as oncogenes. However, YAP might also play tumor-suppressing role. For example, YAP can bind to DNA-binding tumor suppressors including RUNXs and p73. Thus, YAP might act either as an oncogene or tumor suppressor depending on its binding partners. Here, we summarize roles of YAP depending on its DNA-binding partners and discuss context-dependent functions of YAP/TAZ. [BMB Reports 2018; 51(3): 126-133].

Runx3 inactivation is a crucial early event in the development of lung adenocarcinoma.

Targeted inactivation of Runx3 in mouse lung induced mucinous and nonmucinous adenomas and markedly shortened latency of adenocarcinoma formation induced by oncogenic K-Ras. RUNX3 was frequently inactivated in K-RAS mutated human lung adenocarcinomas. A functional genetic screen of a fly mutant library and molecular analysis in cultured cell lines revealed that Runx3 forms a complex with BRD2 in a K-Ras-dependent manner in the early phase of the cell cycle; this complex induces expression of p14(ARF)/p19(Arf) and p21(WAF/CIP). When K-Ras was constitutively activated, the Runx3-BRD2 complex was stably maintained and expression of both p14(ARF) and p21(WAF/CIP) was prolonged. These results provide a missing link between oncogenic K-Ras and the p14(ARF)-p53 pathway, and may explain how cells defend against oncogenic K-Ras.

Src kinase phosphorylates RUNX3 at tyrosine residues and localizes the protein in the cytoplasm.

RUNX3 is a transcription factor that functions as a tumor suppressor. In some cancers, RUNX3 expression is down-regulated, usually due to promoter hypermethylation. Recently, it was found that RUNX3 can also be inactivated by the mislocalization of the protein in the cytoplasm. The molecular mechanisms controlling this mislocalization are poorly understood. In this study, we found that the overexpression of Src results in the tyrosine phosphorylation and cytoplasmic localization of RUNX3. We also found that the tyrosine residues of endogenous RUNX3 are phosphorylated and that the protein is localized in the cytoplasm in Src-activated cancer cell lines. We further showed that the knockdown of Src by small interfering RNA, or the inhibition of Src kinase activity by a chemical inhibitor, causes the re-localization of RUNX3 to the nucleus. Collectively, our results demonstrate that the tyrosine phosphorylation of RUNX3 by activated Src is associated with the cytoplasmic localization of RUNX3 in gastric and breast cancers.

Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3.

Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27(Kip1), and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.