RESUMO
Smad3 linker phosphorylation is a candidate target for several kinases that play important roles in cancer cell initiation, proliferation and progression. Also, Smad3 is an essential intracellular mediator of TGF-ß1-induced transcriptional responses during carcinogenesis. Therefore, it is highly advantageous to identify and develop inhibitors targeting Smad3 linker phosphorylation for the treatment of cancers. Galangin (3,5,7-trihydroxyflavone) has been known to be an active flavonoid showing a cytotoxic effect on several cancer cells. However, the mechanism of action of galangin in various cancers remains unclear, and there has been no report concerning regulation of Smad3 phosphorylation by galangin. In the present study, we show that galangin significantly induced apoptosis and inhibited cell proliferation in the presence of TGF-ß1 in both human prostate and pancreatic cancer cell lines. Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of Smad3 linker region.
Assuntos
Proliferação de Células/efeitos dos fármacos , Flavonoides/administração & dosagem , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Treonina/metabolismo , Resultado do TratamentoRESUMO
The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.
Assuntos
Amelogênese , Proteínas de Homeodomínio , Camundongos Knockout , Células-Tronco , Fatores de Transcrição , Animais , Humanos , Camundongos , Amelogênese/genética , Diferenciação Celular/genética , Epitélio/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Via de Sinalização WntRESUMO
BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.
Assuntos
Doenças Ósseas , Reabsorção Óssea , Osteoporose , Tetraspaninas , Animais , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Integrina alfaVbeta3/metabolismo , Osteoclastos , Osteoporose/genética , Osteoporose/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.
Assuntos
Células-Tronco Germinativas Adultas , Proteínas Associadas aos Microtúbulos , Animais , Camundongos , Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/fisiologia , Ciclo Celular/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , MasculinoRESUMO
Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-ß and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-ß1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3ß and subsequent Smurf1 recruitment, promoted ß-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4-/- mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.
Assuntos
Osso e Ossos , Cartilagem , Células-Tronco Mesenquimais , Proteínas Associadas aos Microtúbulos , Proteínas Serina-Treonina Quinases , Animais , Feminino , Camundongos , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Cartilagem/citologia , Cartilagem/metabolismo , Diferenciação Celular/genética , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Osteogênese/genética , Proteínas Serina-Treonina Quinases/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização WntRESUMO
Spermatogenesis is an important cellular differentiation process that produces the male gametes and remains active throughout the individual's lifespan. Sertoli cell-only syndrome (SCO) refers to the dysfunction of the male reproductive system, including infertility. Accurate self-renewal of spermatogonial stem cells (SSCs) is essential to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice and MAST4 expression shifted to Leydig cells and spermatids throughout puberty. Mast4 knockout (KO) testes were reduced in size compared to wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. In addition, MAST4 phosphorylated the Ets-related molecule (ERM), specifically the serine 367 residue. The phosphorylation of ERM ultimately controls the transcription of ERM target genes related to SSC self-renewal. The expression of spermatogenesis-associated proteins was significantly decreased whereas Sertoli cell markers were increased in Mast4 KO testes, which was well-founded by RNA-sequencing analysis. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway and this association helps us explore the capacity of SSCs to maintain a vertebrate stem cell niche.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espermatogônias/fisiologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos KnockoutRESUMO
Although tetraarsenic hexoxide is known to exert an anti-tumor effect by inducing apoptosis in various cancer cells, its effect on other forms of regulated cell death remains unclear. Here, we show that tetraarsenic hexoxide induces the pyroptotic cell death through activation of mitochondrial reactive oxygen species (ROS)-mediated caspase-3/gasdermin E (GSDME) pathway, thereby suppressing tumor growth and metastasis of triple-negative breast cancer (TNBC) cells. Interestingly, tetraarsenic hexoxide-treated TNBC cells exhibited specific pyroptotic characteristics, including cell swelling, balloon-like bubbling, and LDH releases through pore formation in the plasma membrane, eventually suppressing tumor formation and lung metastasis of TNBC cells. Mechanistically, tetraarsenic hexoxide markedly enhanced the production of mitochondrial ROS by inhibiting phosphorylation of mitochondrial STAT3, subsequently inducing caspase-3-dependent cleavage of GSDME, which consequently promoted pyroptotic cell death in TNBC cells. Collectively, our findings highlight tetraarsenic hexoxide-induced pyroptosis as a new therapeutic strategy that may inhibit cancer progression of TNBC cells.
Assuntos
Antineoplásicos/farmacologia , Trióxido de Arsênio/farmacologia , Caspase 3/metabolismo , Mitocôndrias/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Caspase 3/genética , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. METHODS: We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. RESULTS: In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-ß1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. CONCLUSIONS: These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers.
RESUMO
[This corrects the article on p. 1 in vol. 23, PMID: 29629343.].