RESUMO
Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.
Assuntos
Fertilidade , Hexoquinase , Infertilidade Masculina , Camundongos Knockout , Capacitação Espermática , Espermatozoides , Tirosina , Animais , Hexoquinase/genética , Hexoquinase/metabolismo , Masculino , Camundongos , Fosforilação , Espermatozoides/metabolismo , Capacitação Espermática/genética , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Fertilidade/genética , Tirosina/metabolismo , Feminino , Testículo/metabolismo , Motilidade dos Espermatozoides/genética , Glicólise , Espermatogênese/genéticaRESUMO
Recent studies showed that both prostaglandin E2 (PGE2) and transient receptor potential melastatin 7 (TRPM7) play important roles in migration and proliferation of human glioblastoma cells. In this study, we tested the association between PGE2 and TRPM7. We found that PGE2 increased TRPM7 currents in HEK293 and human glioblastoma A172 cells. The PGE2 EP3 receptor antagonist L-798106 abrogated the PGE2 stimulatory effect, while EP3 agonist 17-phenyl trinor prostaglandin E2 (17-pt-PGE2) mimicked the effect of PEG2 on TRPM7. The TRPM7 phosphotransferase activity-deficient mutation, K1646R had no effect on PGE2 induced increase of TRPM7 currents. Inhibition of protein kinase A (PKA) activity by Rp-cAMP increased TRPM7 currents. TRPM7 PKA phosphorylation site mutation S1269A abolished the PGE2 effect on TRPM7 currents. PGE2 increased both mRNA and membrane protein expression of TRPM7 in A172 cells. Knockdown of TRPM7 by shRNA abrogated the PGE2 stimulated migration and proliferation of A172 cells. Blockage of TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) or NS8593 had a similar effect as TRPM7-shRNA. In conclusion, our results demonstrate that PGE2 activates TRPM7 via EP3/PKA signalling pathway, and that PGE2 enhances migration and proliferation of human glioblastoma cells by up-regulation of the TRPM7 channel.
Assuntos
Dinoprostona/genética , Glioblastoma/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Prostaglandina E Subtipo EP3/genética , Canais de Cátion TRPM/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Células HEK293 , Humanos , Mutação/genética , Fosforilação/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores de Prostaglandina E Subtipo EP3/agonistas , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) ranks as the second-leading cause of cancer-related death worldwide with metastases being the main cause of cancer-related death. Here, we investigated the genomic and transcriptomic alterations in matching adjacent normal tissues, primary tumors, and metastatic tumors of CRC patients. METHODS: We performed whole genome sequencing (WGS), multi-region whole exome sequencing (WES), simultaneous single-cell RNA-Seq, and single-cell targeted cDNA Sanger sequencing on matching adjacent normal tissues, primary tumors, and metastatic tumors from 12 metastatic colorectal cancer patients (n=84 for genomes, n=81 for exomes, n=9120 for single cells). Patient-derived tumor organoids were used to estimate the anti-tumor effects of a PPAR inhibitor, and self-renewal and differentiation ability of stem cell-like tumor cells. RESULTS: We found that the PPAR signaling pathway was prevalently and aberrantly activated in CRC tumors. Blocking of PPAR pathway both suppressed the growth and promoted the apoptosis of CRC organoids in vitro, indicating that aberrant activation of the PPAR signaling pathway plays a critical role in CRC tumorigenesis. Using matched samples from the same patient, distinct origins of the metastasized tumors between lymph node and liver were revealed, which was further verified by both copy number variation and mitochondrial mutation profiles at single-cell resolution. By combining single-cell RNA-Seq and single-cell point mutation identification by targeted cDNA Sanger sequencing, we revealed important phenotypic differences between cancer cells with and without critical point mutations (KRAS and TP53) in the same patient in vivo at single-cell resolution. CONCLUSIONS: Our data provides deep insights into how driver mutations interfere with the transcriptomic state of cancer cells in vivo at a single-cell resolution. Our findings offer novel knowledge on metastatic mechanisms as well as potential markers and therapeutic targets for CRC diagnosis and therapy. The high-precision single-cell RNA-seq dataset of matched adjacent normal tissues, primary tumors, and metastases from CRCs may serve as a rich resource for further studies.
Assuntos
Neoplasias Colorretais , Metástase Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , DNA Complementar , Genômica , Humanos , Mutação , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Receptores Ativados por Proliferador de Peroxissomo/genética , TranscriptomaRESUMO
In this study, we tested the effect of melatonin on proliferation and differentiation of neural stem/progenitor cells (NSPCs) obtained from adult mouse spinal cord. We found that melatonin increases neurosphere formation from adult spinal cord NSPCs but does not alter the differentiation of the cells. Western blot results show that adult spinal cord NSPCs express both MT1 and MT2 melatonin receptors. The melatonin receptor antagonist 4P-PDOT abrogates the melatonin-induced neurosphere formation. Melatonin increases the phosphorylation level of protein kinase B (AKT). Blockage of phosphatidylinositol 3-kinase (PI3K), a kinase upstream of AKT, abolishes the stimulatory effect of melatonin on spinal cord NSPCs. We conclude that melatonin promotes the proliferation of adult spinal cord NSPCs via the PI3K/AKT signaling pathway.