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World Journal of Gastroenterology May 2024Cell division cyclin 25C () is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research...
BACKGROUND
Cell division cyclin 25C () is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research has shown that could be a potential therapeutic target for cancers, particularly for hepatocellular carcinoma (HCC). However, the specific regulatory mechanisms underlying the role of in HCC tumorigenesis and development remain incompletely understood.
AIM
To explore the impact of on cell proliferation and apoptosis, as well as its regulatory mechanisms in HCC development.
METHODS
Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences (LV- shRNA) to knock down . Subsequently, a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into mice to assess the effects of knockdown on HCC development . Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays, respectively. The expression of endoplasmic reticulum (ER) stress-related molecules (glucose-regulated protein 78, X-box binding protein-1, and C/EBP homologous protein) was measured in both cells and subcutaneous xenografts using quantitative real-time PCR (qRT-PCR) and western blotting. Additionally, apoptosis was investigated using flow cytometry, qRT-PCR, and western blotting.
RESULTS
was stably suppressed in Hepa1-6 and B16 cells through LV- shRNA transduction. A xenograft model with knockdown was successfully established and that downregulation of expression significantly inhibited HCC growth in mice. knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response, ultimately promoting ER stress-induced apoptosis in HCC cells.
CONCLUSION
The regulatory mechanism of in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.
Topics: Animals; Endoplasmic Reticulum Stress; Carcinoma, Hepatocellular; Cell Proliferation; cdc25 Phosphatases; Apoptosis; Liver Neoplasms; Cell Line, Tumor; Mice; Gene Knockdown Techniques; Cell Movement; Mice, Inbred C57BL; Humans; RNA, Small Interfering; Male; Gene Expression Regulation, Neoplastic; Xenograft Model Antitumor Assays; Carcinogenesis
PubMed: 38817663
DOI: 10.3748/wjg.v30.i19.2564 -
Neuroreport Jul 2024Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive...
Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.
Topics: Astrocytes; Connexin 43; Animals; Phosphorylation; Calcineurin; Rats; Cell Membrane Permeability; Cells, Cultured; Rats, Sprague-Dawley
PubMed: 38813906
DOI: 10.1097/WNR.0000000000002051 -
BMC Biology May 2024The innate immune system serves as the first line of host defense. Transforming growth factor-β-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell...
BACKGROUND
The innate immune system serves as the first line of host defense. Transforming growth factor-β-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease.
RESULTS
In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation.
CONCLUSIONS
Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.
Topics: Receptor-Interacting Protein Serine-Threonine Kinases; Humans; Phosphoprotein Phosphatases; MAP Kinase Kinase Kinases; Necroptosis; Immunity, Innate
PubMed: 38807188
DOI: 10.1186/s12915-024-01901-5 -
Open Biology May 2024The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The...
The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.
Topics: Aurora Kinase B; Phosphorylation; Humans; Histones; Mitosis; Protein Phosphatase 1; Cell Cycle Proteins; HeLa Cells; Spindle Apparatus; Centromere; Nuclear Proteins
PubMed: 38806145
DOI: 10.1098/rsob.230460 -
Cell Reports Jun 2024Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1 DNA damage response (DDR). We study DDR and NER in aging cells and find that...
Survival from UV-induced DNA lesions relies on nucleotide excision repair (NER) and the Mec1 DNA damage response (DDR). We study DDR and NER in aging cells and find that old cells struggle to repair DNA and activate Mec1. We employ pharmacological and genetic approaches to rescue DDR and NER during aging. Conditions activating Snf1 rescue DDR functionality, but not NER, while inhibition of the TORC1-Sch9 axis restores NER and enhances DDR by tuning PP2A activity, specifically in aging cells. Age-related repair deficiency depends on Snf1-mediated phosphorylation of Sch9 on Ser160 and Ser163. PP2A activity in old cells is detrimental for DDR and influences NER by modulating Snf1 and Sch9. Hence, the DDR and repair pathways in aging cells are influenced by the metabolic tuning of opposing AMPK and TORC1 networks and by PP2A activity. Specific Sch9 phospho-isoforms control DDR and NER efficiency, specifically during aging.
Topics: DNA Repair; DNA Damage; Saccharomyces cerevisiae Proteins; Cellular Senescence; Phosphorylation; Saccharomyces cerevisiae; Protein Serine-Threonine Kinases; Protein Phosphatase 2; Intracellular Signaling Peptides and Proteins; Aging
PubMed: 38805395
DOI: 10.1016/j.celrep.2024.114281 -
Life Science Alliance Aug 2024FK506-binding protein 52 (FKBP52) is a member of the FKBP family of proline isomerases. FKBP52 is up-regulated in various cancers and functions as a positive regulator...
FK506-binding protein 52 (FKBP52) is a member of the FKBP family of proline isomerases. FKBP52 is up-regulated in various cancers and functions as a positive regulator of steroid hormone receptors. Depletion of FKBP52 is known to inhibit cell proliferation; however, the detailed mechanism remains poorly understood. In this study, we found that FKBP52 depletion decreased transcription, leading to stabilization of p53, and suppressed cell proliferation. We identified NFATc1 and NFATc3 as transcription factors that regulate We also found that FKBP52 associated with NFATc3 and facilitated its nuclear translocation. In addition, calcineurin, a well-known Ca phosphatase essential for activation of NFAT, plays a role in transcription. Supporting this notion, expression was found to be regulated by intracellular Ca Taken together, these findings reveal a new role of FKBP52 in promoting cell proliferation via the NFAT-MDM2-p53 axis, and indicate that inhibition of FKBP52 could be a new therapeutic tool to activate p53 and inhibit cell proliferation.
Topics: Humans; Tumor Suppressor Protein p53; Tacrolimus Binding Proteins; Cell Proliferation; NFATC Transcription Factors; Proto-Oncogene Proteins c-mdm2; Cell Line, Tumor; Calcium; Calcineurin; Gene Expression Regulation, Neoplastic; Neoplasms; Signal Transduction
PubMed: 38803221
DOI: 10.26508/lsa.202302426 -
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... May 2024Objective To explore the regulatory role of dual-specificity phosphatase 5 (DUSP5) in BCG-mediated inflammatory response in mouse RAW264.7 macrophages. Methods Western...
Objective To explore the regulatory role of dual-specificity phosphatase 5 (DUSP5) in BCG-mediated inflammatory response in mouse RAW264.7 macrophages. Methods Western blot analysis was employed to detect the expression changes of DUSP5 in BCG-infected RAW264.7 macrophages at the period of 0.5, 1, 2, 4, 6, 8, 12 and 24 hours. Intracellular DUSP5 was reduced by small interfering RNA (siRNA) and transfected RAW264.7 macrophages were divided into siRNA-negative control (si-NC) group, DUSP5 knockdown (si-DUSP5) group, si-NC combined BCG infection group, and si-DUSP5 combined BCG infection group. Real-time quantitative PCR was conducted to measure the mRNA expression of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and IL-10 in cells. ELISA was performed to measure the concentration of the cytokines in cell culture medium. Western blot analysis was performed to detect the expression changes of cellular nuclear factor κB (NF-κB) and phosphorylated NF-κB (p-NF-κB). Results BCG infection upregulated DUSP5 protein expression in RAW264.7 macrophages with the expression of DUSP5 reaching the peak after 4 hours' BCG stimulation. Comparing with si-NC combined BCG infection group, DUSP5 knockdown inhibited the expression and secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, while the expression of the anti-inflammatory factor IL-10 was not affected by DUSP5. Moreover, knockdown of DUSP5 inhibited the phosphorylation of NF-κB in cells. Conclusion DUSP5 knockdown inhibites BCG-mediated macrophage inflammatory response via blocking NF-κB signaling activation.
Topics: Animals; Mice; RAW 264.7 Cells; Dual-Specificity Phosphatases; NF-kappa B; Signal Transduction; Macrophages; Inflammation; Gene Knockdown Techniques; Mycobacterium bovis; Cytokines
PubMed: 38790096
DOI: No ID Found -
Molecular Cancer Research : MCR May 2024HBV-associated hepatocellular carcinoma (HCC) represents the prevalent form of HCC, with HBx protein being a crucial oncoprotein. Numerous members of the protein...
HBV-associated hepatocellular carcinoma (HCC) represents the prevalent form of HCC, with HBx protein being a crucial oncoprotein. Numerous members of the protein tyrosine phosphatase non-receptor (PTPN) family have been confirmed to be significantly associated with the occurrence and progression of malignant tumors. Our group has previously identified the involvement of PTPN13 in HCC. However, the roles of other PTPNs in HCC still requires further investigation. In this study, we found PTPN18 expression was significantly downregulated within HCC tissues compared to that in adjacent non-tumor tissues and normal liver tissues. Functionally, PTPN18 exerted inhibitory effects on the proliferation, migration, invasion, and sphere-forming capability of HCC cells, while concurrently promoting apoptotic processes. Through phospho-protein microarray screening followed by subsequent validation experiments, we identified that PTPN18 could activate the p53 signaling pathway and suppress the AKT/FOXO1 signaling cascade in HCC cells. Moreover, we found that the HBx protein mediated the repression of PTPN18 expression by upregulating miR-128-3p. Collectively, our study unveiled the role of PTPN18 as a tumor suppressor in HBV-related HCC. Implications: Our findings revealed PTPN18 might serve as a potential diagnostic and therapeutic target for HBV-related HCC.
PubMed: 38787319
DOI: 10.1158/1541-7786.MCR-23-0696 -
European Journal of Cell Biology Jun 2024The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation...
The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation of the retinal pigmented epithelium (RPE), and in humans, some mutations in MITF induce congenital eye malformations. Herein, we explore the function and regulation of Mitf in Drosophila eye development and uncover two roles. We find that knockdown of Mitf results in retinal displacement (RDis), a phenotype associated with abnormal eye formation. Mitf functions in the peripodial epithelium (PE), a retinal support tissue akin to the RPE, to suppress RDis, via the Hippo pathway effector Yorkie (Yki). Yki physically interacts with Mitf and can modify its transcriptional activity in vitro. Severe loss of Mitf, instead, results in the de-repression of retinogenesis in the PE, precluding its development. This activity of Mitf requires the protein phosphatase 2 A holoenzyme STRIPAK-PP2A, but not Yki; Mitf transcriptional activity is potentiated by STRIPAK-PP2A in vitro and in vivo. Knockdown of STRIPAK-PP2A results in cytoplasmic retention of Mitf in vivo and in its decreased stability in vitro, highlighting two potential mechanisms for the control of Mitf function by STRIPAK-PP2A. Thus, Mitf functions in a context-dependent manner as a key determinant of form and fate in the Drosophila eye progenitor epithelium.
Topics: Animals; Drosophila Proteins; Microphthalmia-Associated Transcription Factor; YAP-Signaling Proteins; Trans-Activators; Nuclear Proteins; Eye; Protein Phosphatase 2; Drosophila melanogaster; Epithelium; Cell Differentiation; Homeodomain Proteins
PubMed: 38776620
DOI: 10.1016/j.ejcb.2024.151421 -
Proceedings of the National Academy of... May 2024C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane...
C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.
Topics: Humans; Crystallography, X-Ray; Endoplasmic Reticulum; Intracellular Membranes; Membrane Proteins; Phosphoprotein Phosphatases; Protein Binding
PubMed: 38776370
DOI: 10.1073/pnas.2321167121