RESUMO
Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent ß-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of ß-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, ß-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.
Assuntos
Benzeno/toxicidade , Citocromo P-450 CYP2E1/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Citocromo P-450 CYP2E1/genética , Células Hep G2 , Humanos , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/genéticaRESUMO
Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein ß-catenin. Epithelial accumulation of ß-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or ß-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of ß-catenin/FOXO1 signaling pathway.
Assuntos
Células Epiteliais/patologia , Fibroblastos/patologia , Fibrose/patologia , Nefropatias/patologia , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Animais , Apoptose , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Nefropatias/etiologia , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Transdução de SinaisRESUMO
The four R-spondins (RSPO1-4) strongly potentiate Wnt signaling and play critical roles in normal development, adult stem cell survival, and cancer development and aggressiveness. All four RSPOs have been suggested to potentiate Wnt signaling by binding to three related receptors, i.e. leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6), and then inducing the clearance of two E3 ubiquitin ligases (RNF43 and ZNRF3) that otherwise would ubiquitinate Wnt receptors for degradation. Here, we show that RSPO1-4 have differential dependence on LGRs in potentiating Wnt/ß-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/ß-catenin signaling response to RSPO1 and RSPO4 and strongly impaired the response to RSPO3. RSPO2, however, retained robust activity albeit with decreased potency. Complete rescue of RSPO1-4 activity in LGR4KO cells required the seven-transmembrane domain of LGR4. Furthermore, an RSPO2 mutant with normal binding affinity to ZNRF3 but no or little binding to LGR4 or LGR5 still potentiated Wnt/ß-catenin signaling in vitro, supported the growth of intestinal organoids ex vivo, and stimulated intestinal crypt growth in vivo Mechanistically, RSPO2 could increase Wnt receptor levels in the absence of any LGR without affecting ZNRF3 endocytosis and stability. These findings suggest that RSPO1-4 use distinct mechanisms in regulating Wnt and other signaling pathways, which have important implications for understanding the pleiotropic functions of RSPOs and LGRs in both normal and cancer development.
Assuntos
Transdução de Sinais , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organoides/citologia , Organoides/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
Cellular FLICE-like inhibitory protein (c-FLIPL) is a key inhibitory protein in the extrinsic apoptotic pathway. Recent studies showed that c-FLIPL could translocate into the nucleus and might be involved in the Wnt signaling pathway. The nuclear function of c-FLIPL was still unclear. Here we found a novel c-FLIPL-associated protein TIP49, which is a nuclear protein identified as a cofactor in the transcriptional regulation of ß-catenin. They had co-localization in the nucleus and the DED domain of c-FLIPL was required for the association with TIP49. By performing ChIP experiments, C-FLIPL was detected in the ITF-2 locus and facilitated TIP49 accumulation in the formation of complexes at the T-cell-specific transcription factor site of human ITF-2 promoter. When TIP49 knockdown, c-FLIPL-driven Wnt activation, and cell proliferation were inhibited, suggesting that a role of nuclear c-FLIPL involved in modulation of the Wnt pathway was in a TIP49-dependent manner. Elevated expression of c-FLIPL and TIP49 that coincided in human lung cancers were analyzed in silico using the Oncomine database. Their high expressions were reconfirmed in six lung cancer cell lines and correlated with cell growth. The association of c-FLIPL and TIP49 provided an additional mechanism involved in c-FLIPL-mediated functions, including Wnt activation.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteínas de Transporte/genética , Divisão Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA Helicases/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ß-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ß-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate ß-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ß-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteína Axina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Tanquirases/metabolismo , beta Catenina/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteína Axina/genética , Western Blotting , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Imunofluorescência , Células HCT116 , Humanos , Masculino , Ligação Proteica , Especificidade por Substrato , Tanquirases/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Técnicas do Sistema de Duplo-Híbrido , beta Catenina/genéticaRESUMO
Hematopoietic and leukemic stem cells (HSCs and LSCs) have self-renewal ability to maintain normal hematopoiesis and leukemia propagation, respectively. Tcf1 and Lef1 transcription factors are expressed in HSCs, and targeting both factors modestly expanded the size of the HSC pool due to diminished HSC quiescence. Functional defects of Tcf1/Lef1-deficient HSCs in multi-lineage blood reconstitution was only evident under competitive conditions or when subjected to repeated regenerative stress. These are mechanistically due to direct positive regulation of Egr and Tcf3 by Tcf1 and Lef1, and significantly, forced expression of Egr1 in Tcf1/Lef1-deficient HSCs restored HSC quiescence. In a preclinical CML model, loss of Tcf1/Lef1 did not show strong impact on leukemia initiation and progression. However, when transplanted into secondary recipients, Tcf1/Lef1-deficient LSCs failed to propagate CML. By induced deletion of Tcf1 and Lef1 in pre-established CML, we further demonstrated an intrinsic requirement for these factors in LSC self-renewal. When combined with imatinib therapy, genetic targeting of Tcf1 and Lef1 potently diminished LSCs and conferred better protection to the CML recipients. LSCs are therefore more sensitive to loss of Tcf1 and Lef1 than HSCs in their self-renewal capacity. The differential requirements in HSCs and LSCs thus identify Tcf1 and Lef1 transcription factors as novel therapeutic targets in treating hematological malignancies, and inhibition of Tcf1/Lef1-regulated transcriptional programs may thus provide a therapeutic window to eliminate LSCs with minimal side effect on normal HSC functions.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Leucemia/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Células-Tronco Hematopoéticas/patologia , Fator 1-alfa Nuclear de Hepatócito/genética , Leucemia/genética , Leucemia/patologia , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologiaRESUMO
Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of ß-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential ß-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific ß-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/ß-catenin but not CBP/ß-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/ß-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/ß-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/ß-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting ß-catenin to modulate adult progenitor cell behavior in disease.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Proteína p300 Associada a E1A/fisiologia , Proteína Quinase C/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/fisiologia , Células Epiteliais Alveolares/fisiologia , Animais , Aquaporina 5/genética , Aquaporina 5/metabolismo , Linhagem Celular , Impedância Elétrica , Expressão Gênica , Camundongos , Camundongos Knockout , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Ratos , Via de Sinalização Wnt , Proteína Wnt-5aRESUMO
The A-kinase anchoring protein (AKAP) GSK3ß interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3ß (GSK3ß). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3ß are required for the regulation of ß-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets ß-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause ß-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the ß-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3ß facilitates control of the destabilizing phosphorylation of ß-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on ß-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with ß-catenin. The regulation of ß-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3ß, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3ß is a conserved GSK3ß interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3ß by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Proteínas de Ancoragem à Quinase A , Células A549 , Proteínas Quinases Dependentes de AMP Cíclico/genética , Glicogênio Sintase Quinase 3 beta/genética , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Proteínas Repressoras/genética , beta Catenina/genéticaRESUMO
Histone deacetylases (Hdacs) regulate endochondral ossification by suppressing gene transcription and modulating cellular responses to growth factors and cytokines. We previously showed that Hdac7 suppresses Runx2 activity and osteoblast differentiation. In this study, we examined the role of Hdac7 in postnatal chondrocytes. Hdac7 was highly expressed in proliferating cells within the growth plate. Postnatal tissue-specific ablation of Hdac7 with a tamoxifen-inducible collagen type 2a1-driven Cre recombinase increased proliferation and ß-catenin levels in growth plate chondrocytes and expanded the proliferative zone. Similar results were obtained in primary chondrocyte cultures where Hdac7 was deleted with adenoviral-Cre. Hdac7 bound ß-catenin in proliferating chondrocytes, but stimulation of chondrocyte maturation promoted the translocation of Hdac7 to the cytoplasm where it was degraded by the proteasome. As a result, ß-catenin levels and transcription activity increased in the nucleus. These data demonstrate that Hdac7 suppresses proliferation and ß-catenin activity in chondrocytes. Reducing Hdac7 levels in early chondrocytes may promote the expansion and regeneration of cartilage tissues.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Lâmina de Crescimento/metabolismo , Histona Desacetilases/genética , beta Catenina/genética , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Condrócitos/citologia , Citoplasma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Lâmina de Crescimento/citologia , Lâmina de Crescimento/crescimento & desenvolvimento , Histona Desacetilases/deficiência , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Proteólise , Transdução de Sinais , beta Catenina/agonistas , beta Catenina/metabolismoRESUMO
The Wnt signalling pathway plays key roles in cell proliferation, differentiation and fate decisions in embryonic development and maintenance of adult tissues, and the twelve Armadillo (ARM) repeat-containing protein ß-catenin acts as the signal transducer in this pathway. Here we investigate the interaction between ß-catenin's ARM repeat domain and the intrinsically disordered protein adenomatous polyposis coli (APC). APC is a giant multivalent scaffold that brings together the different components of the so-called "ß-catenin destruction complex", which drives ß-catenin degradation via the ubiquitin-proteasome pathway. Mutations and truncations in APC, resulting in loss of APC function and hence elevated ß-catenin levels and upregulation of Wnt signalling, are associated with numerous cancers including colorectal carcinomas. APC has a long intrinsically disordered region (IDR) that contains a series of 15-residue and 20-residue binding regions for ß-catenin. Here we explore the multivalent nature of the interaction of ß-catenin with the highest affinity APC repeat, both at equilibrium and under kinetic conditions. We use a combination of single-site substitutions, deletions and insertions to dissect the mechanism of molecular recognition and the roles of the three ß-catenin-binding subdomains of APC.
RESUMO
Dendritic cells (DCs) control the strength and quality of antigen-specific adaptive immune responses. This is critical for launching a robust immunity against invading pathogens while maintaining a state of tolerance to self-antigens. However, this also represents a fundamental barrier to anti-tumor immune responses and cancer immunotherapy. DCs in the tumor microenvironment (TME) play a key role in this process. The factors in the TME and signaling networks that program DCs to a regulatory state are not fully understood. Recent advances point to novel mechanisms by which the canonical Wnt signaling cascade in DCs regulates immune suppression, and the same pathway in tumors is associated with the evasion of anti-tumor immunity. Here, we review these recent advances in the context of the pleiotropic effects of the Wnts in shaping anti-tumor immune responses by modulating DC functions. In addition, we will discuss how Wnt/ß-catenin pathway in DCs can be targeted for successful cancer immunotherapy.