-
Reports of Biochemistry & Molecular... Jan 2021Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of...
BACKGROUND
Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of amyloid-beta peptides, play a pivotal role in AD pathophysiology. This study focuses on the evaluation of the effects of amyloid precursor protein (APP) overexpression on NF-κB, Rho-GTPase and Bcl-2 mediated pro-apoptotic pathways in neuronal cells.
METHODS
A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells.
RESULTS
In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group.
CONCLUSION
APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.
PubMed: 33969135
DOI: 10.52547/rbmb.9.4.417 -
International Journal of Molecular... Oct 2020Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in...
Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with and mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 , either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by HO caused a dose-dependent increase in gene and CCN3 protein expression, along with enhanced expression of and mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced promoter activity via . Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of and mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.
Topics: Animals; Cartilage, Articular; Cell Line, Tumor; Cells, Cultured; Cellular Senescence; Chondrocytes; Cyclin-Dependent Kinase Inhibitor p21; Humans; Mice; Mice, Inbred C57BL; Nephroblastoma Overexpressed Protein; Osteoarthritis, Knee; Rats; Tumor Suppressor Protein p53
PubMed: 33066270
DOI: 10.3390/ijms21207556 -
Blood Advances Aug 2021Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point...
Although ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal-related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2-/-γc-/- mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non-BTK-targeted therapeutics.
Topics: Adenine; Agammaglobulinaemia Tyrosine Kinase; Animals; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Piperidines; Pyrazoles; Pyrimidines
PubMed: 34424317
DOI: 10.1182/bloodadvances.2020003821 -
Journal of B.U.ON. : Official Journal... 2021To explore whether vasorin protein (VASN) can affect the proliferation of laryngeal cancer cells through the regulation of yes-associated protein (YAP)/TAZ...
PURPOSE
To explore whether vasorin protein (VASN) can affect the proliferation of laryngeal cancer cells through the regulation of yes-associated protein (YAP)/TAZ (transcriptional co-activator with PDZ binding motif), and then promote the development of laryngeal cancer.
METHODS
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of VASN in laryngeal carcinoma tissues and different T-stage tumor patients, and the correlation between VASN expression and clinicopathological features was analyzed. The diagnostic value of VASN for laryngeal cancer was assessed by receiver operating characteristic (ROC) analysis. Kaplan-Meier method was used to plot the survival curves of patients with different VASN expression levels. After knocking down VASN in Hep-2 cells or in overexpressing VASN in TU212 cells, cell viability, proliferation ability and protein expression level of YAP/TAZ were detected by cell counting kit-8 (CCK-8), plate cloning assay and Western blot. Furthermore, YAP was overexpressed or knocked down simultaneously to evaluate its effect on the viability and proliferation ability of cells.
RESULTS
The expression of VASN in laryngeal carcinoma was significantly higher than that in the normal control group, while, at the same time, the expression of VASN in the t3+t4 tumor patients was significantly higher than that in the t1+t2 tumors. We also found that the expression level of VASN was closely related to N stage, T stage, and lymph node metastasis, suggesting that VASN had a certain diagnostic value for laryngeal cancer. After knocking down VASN in cells, the cell viability, proliferative capacity and YAP/TAZ protein expression level decreased significantly. Besides, overexpressing YAP could reverse the inhibition of cell viability and proliferation ability caused by VASN knockdown.
CONCLUSIONS
VASN can promote the development of laryngeal cancer by affecting the expression of YAP/TAZ.
Topics: Carrier Proteins; Cell Proliferation; Humans; Laryngeal Neoplasms; Membrane Proteins; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Tumor Cells, Cultured; YAP-Signaling Proteins
PubMed: 34565020
DOI: No ID Found -
Histochemistry and Cell Biology Nov 2021Elevated expression of the nucleoporin Nup88, a constituent of the nuclear pore complex, is seen in various types of malignant tumors, but whether this overexpression...
Elevated expression of the nucleoporin Nup88, a constituent of the nuclear pore complex, is seen in various types of malignant tumors, but whether this overexpression contributes to the malignant phenotype has yet to be determined. Here, we investigated the effect of the overexpression of Nup88 on the migration and invasion of cervical cancer HeLa cells. The overexpression of Nup88 promoted a slight but significant increase in both migration and invasion, whereas knockdown of Nup88 by RNA interference suppressed these phenotypes. The observed phenotypes in Nup88-overexpressing HeLa cells were not due to the progression of the epithelial-to-mesenchymal transition or activation of NF-κB, which are known to be important for cell migration and invasion. Instead, we identified an upregulation of matrix metalloproteinase-12 (MMP-12) at both the gene and protein levels in Nup88-overexpressing HeLa cells. Upregulation of MMP-12 protein by the overexpression of Nup88 was also observed in one other cervical cancer cell line and two prostate cancer cell lines but not 293 cells. Treatment with a selective inhibitor against MMP-12 enzymatic activity significantly suppressed the invasive ability of HeLa cells induced by Nup88 overexpression. Taken together, our results suggest that overexpression of Nup88 can stimulate malignant phenotypes including invasive ability, which is promoted by MMP-12 expression.
Topics: Cell Movement; Cells, Cultured; HeLa Cells; Humans; Nuclear Pore Complex Proteins
PubMed: 34331103
DOI: 10.1007/s00418-021-02020-w -
Bioengineered Apr 2022Osteoarthritis (OA) is a degenerative joint disease that affects cartilage and its peripheral tissues. Up-regulation of Calcium-binding protein 39 (CAB39) has a...
Calcium-binding protein 39 overexpression promotes macrophages from 'M1' into 'M2' phenotype and improves chondrocyte damage in osteoarthritis by activating the AMP-activated protein kinase/sirtuin 1 axis.
Osteoarthritis (OA) is a degenerative joint disease that affects cartilage and its peripheral tissues. Up-regulation of Calcium-binding protein 39 (CAB39) has a significant protective effect on osteoblasts, but the role and related molecular mechanisms of CAB39 in OA have not yet been reported. CAB39 overexpression and knockdown models were set up in chondrocytes (ATDC5) and macrophages (RAW264.7). The OA cell model was induced in ATDC5 cells with IL-1β (10 ng/mL). Cell viability was tested by the cell counting kit-8 assay, apoptosis was checked by flow cytometry. Western blot was applied for checking the expression of MMP3, MMP13, Aggrecan, the AMPK/Sirt-1 pathway, apoptosis-related proteins (Bax, Bcl-2, and Caspase-3), and macrophage phenotypic markers (CD86, iNOS, CD206, and Arg1). An OA model was constructed in mice, and CAB39 overexpression plasmids were administered to the knee cavity of the OA model mice. As a result, CAB39 was down-regulated in IL-1β-treated chondrocytes and OA mice. Overexpressing CAB39 enhanced ATDC5 cell viability and choked IL-1β-mediated apoptosis. Overexpression of CAB39 boosted the polarization of macrophages from M1-phenotype into M2 phenotype. In addition, overexpressing CAB39 facilitated the AMPK/Sirt-1 pathway activation, and AMPK inhibitors reversed the protective effect of CAB39 overexpression on chondrocytes. Moreover, CAB39 exhibited anti-inflammatory effects in OA mice by activating the AMPK/Sirt-1 pathway. Collectively, overexpressing CAB39 heightened macrophages' M2 polarization and declined chondrocyte injury in OA by activating the AMPK/Sirt-1 pathway. AMPK: AMP-activated protein kinaseArg1: arginase 1Bax: Bcl-2-associated X proteinBcl-2: B-cell lymphoma-2CAB39: Calcium-binding protein 39CM: Conditioned mediumDMM: destabilization of the medial meniscusECM: extracellular matrixELISA: enzyme-linked immunosorbent assayFCM: Flow cytometryIL-1β: interleukin-1βIL-4: interleukin-4IL-6: interleukin-6IL-10: interleukin-10IFN - γ: Interferon-gammaIHC: ImmunohistochemistryiNOS: Inducible nitric oxide synthaseLKB1: liver kinase B1MMP3: Matrix metalloproteinase3MMP13:Matrix metalloproteinase13NF-κB: NF-kappaBOA: OsteoarthritisqRT-PCR: Quantitative reverse transcription-polymerase chain reactionRT: room temperatureSirt-1: sirtuin 1STRAD: STE20-related adaptor alphaWB: Western blot.
Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Calcium-Binding Proteins; Chondrocytes; Interleukin-1beta; Macrophages; Mice; Osteoarthritis; Phenotype; Proto-Oncogene Proteins c-bcl-2; Sirtuin 1
PubMed: 35412939
DOI: 10.1080/21655979.2022.2061289 -
Life Science Alliance Oct 2022Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator...
Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.
Topics: Cytokinesis; GTP-Binding Proteins; GTPase-Activating Proteins; Microfilament Proteins; Mitogen-Activated Protein Kinases; Pheromones; Phosphorylation; RGS Proteins; Receptors, G-Protein-Coupled; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35985794
DOI: 10.26508/lsa.202101245 -
Metabolic Engineering Jul 2021With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an...
With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes - these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.
Topics: Blood Proteins; Fermentation; Fungal Proteins; Heme; Hemoglobins; Humans; Molecular Chaperones; Peroxidases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33984513
DOI: 10.1016/j.ymben.2021.05.002 -
Cancer Science Jan 2022CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic...
CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic alterations involving CD28 have been frequently observed in adult T-cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non-overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86-positive non-neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non-overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.
Topics: Adult; Aged; Aged, 80 and over; B7-1 Antigen; B7-2 Antigen; CD28 Antigens; DNA Copy Number Variations; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia-Lymphoma, Adult T-Cell; Male; Middle Aged; Prognosis; Survival Analysis; Tumor Microenvironment; Up-Regulation
PubMed: 34738707
DOI: 10.1111/cas.15191 -
Archives of Virology Mar 2023Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural...
Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural protein 4B (NS4B) has been reported to induce lipogenesis, but the underlying mechanism is unclear. In this study, western blots were performed to investigate the effect of NS4B protein levels on key effectors of the Hippo and AKT signaling pathways. Yes-associated protein (YAP) and moesin-ezrin-radixin-like protein (Merlin) are effectors of the Hippo pathway. NS4B downregulated Merlin and phosphorylated YAP (p-YAP) protein expression while increasing the expression of the key AKT pathway proteins p-AKT and NF-κB. By observing the levels of AKT pathway proteins when Merlin was overexpressed or silenced, it was determined that Merlin mediates the AKT pathway. We suggest that HCV NS4B may mediate the AKT signaling pathway by inhibiting the Hippo pathway. Lipid droplets were observed in Huh7.5 cells overexpressing NS4B, and they increased significantly in number when Merlin was silenced. Overexpression of NS4B and Merlin silencing enhanced the expression of sterol regulatory element binding proteins (SREBPs), which have been demonstrated to be key regulatory factors controlling fatty acid synthesis. NS4B and Merlin silencing also enhanced the in vitro proliferative capacity of hepatocellular carcinoma cells. In conclusion, NS4B induces lipogenesis via the effect of the Hippo-YAP pathway on the AKT signaling pathway and thereby plays a significant role in the pathogenesis of HCV-associated diseases.
Topics: Humans; Hepacivirus; Hippo Signaling Pathway; Lipogenesis; Neurofibromin 2; Proto-Oncogene Proteins c-akt; Carcinoma, Hepatocellular; Viral Nonstructural Proteins; Hepatitis C; Liver Neoplasms
PubMed: 36920600
DOI: 10.1007/s00705-023-05743-4