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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 -
Bioscience, Biotechnology, and... Nov 2022Angiopoietin-like protein 2 (ANGPTL2) plays versatile roles in various cardiovascular diseases. Its connection to doxorubicin (DOX)-related cardiomyopathy, however,...
Angiopoietin-like protein 2 (ANGPTL2) plays versatile roles in various cardiovascular diseases. Its connection to doxorubicin (DOX)-related cardiomyopathy, however, remains elusive. To determine the role of ANGPTL2, an adeno-associated viral vector was used to overexpress ANGPTL2 in the murine heart 4 weeks before DOX treatment (15 mg/kg). Moreover, mice were injected with adenoviral vectors to knock down ANGPTL2 in the myocardium. Echocardiography and hemodynamics were used to determine the cardiac function. The effect of ANGPTL2 and its downstream target were elucidated by applying molecular and biochemical strategies. We found that ANGPTL2 expression was significantly increased in response to DOX stimulation. Moreover, cardiac-specific ANGPTL2 overexpression exacerbated DOX-related cardiac dysfunction, myocardial apoptosis, and oxidative stress. Mechanistically, ANGPTL2 aggravated DOX-induced cardiac injury via inhibiting the dual specificity phosphatase 1 (DUSP1) pathway and DUSP1 overexpression significantly impeded DOX-induced cardiomyopathy in ANGPTL2-overexpressed mice. Altogether, ANGPTL2 aggravated DOX-related cardiac injury by suppressing the DUSP1 pathway.
Topics: Animals; Mice; Angiopoietin-like Proteins; Apoptosis; Cardiomyopathies; Cardiotoxicity; Doxorubicin; Dual Specificity Phosphatase 1; Myocardium; Myocytes, Cardiac; Oxidative Stress
PubMed: 36107816
DOI: 10.1093/bbb/zbac156 -
G3 (Bethesda, Md.) May 2022The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that...
The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.
Topics: Cysteine; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 35485947
DOI: 10.1093/g3journal/jkac106 -
Bioengineered Apr 2022Post-stroke depression (PSD) seriously affects the normal life of patients. Based on the previous sequencing results, this study selected miR-129-5p as the research...
Post-stroke depression (PSD) seriously affects the normal life of patients. Based on the previous sequencing results, this study selected miR-129-5p as the research object, which was significantly reduced in the PSD model by screening. To clarify the regulatory role of miR-129-5p, this study overexpressed and interfered with miR-129-5p in neuronal cells cultured in vitro, tested its effect on neuronal cell autophagy, and determined expressions of fasciculation and elongation protein zeta-1 (FEZ1), short coiled-coil protein (SCOC), unc-51 like autophagy activating kinase 1 (ULK1) and autophagy cargo receptor (NBR1) autophagy-related proteins. The dual-luciferase reporter system and immunoprecipitation were applied to detect the molecular regulatory mechanism of miR-129-5 and FEZ1, SCOC, ULK1 and NBR1. Findings of the present study revealed that the autophagy of neuronal cells was markedly decreased by overexpressing miR-129-5p ( < 0.05), and expressions of FEZ1, SCOC, ULK1 and NBR1 were substantially reduced ( < 0.05). The dual-luciferase reporter system results indicated that FEZ1, SCOC, ULK1 and NBR1 were all miR-129-5p target genes. Furthermore, immunoprecipitation assay revealed that SCOC, ULK1 and NBR1 could directly bind to the FEZ1 protein. The experiments at an animal level demonstrated that miR-129-5p could effectively alleviate the behavioral indicators of PSD model mice. Taken together, this study testified that SCOC/ULK1/NBR1 proteins could directly bind to FEZ1 to form protein complex, and all of the four proteins FEZ1/SCOC/ULK1/NBR1 were miR-129-5p target genes. miR-129-5p overexpression could effectively restore the behavioral characteristics of model mice, and reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1.
Topics: Adaptor Proteins, Signal Transducing; Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Carrier Proteins; Depression; Humans; Intracellular Signaling Peptides and Proteins; Luciferases; Membrane Proteins; Mice; MicroRNAs; Nerve Tissue Proteins
PubMed: 35435132
DOI: 10.1080/21655979.2022.2059910 -
Development Genes and Evolution Dec 2023Hair follicle growth is cyclical, and hair cycle dysfunction can lead to hair follicle-related disorders, including alopecia and hirsutism. The objective was to...
Hair follicle growth is cyclical, and hair cycle dysfunction can lead to hair follicle-related disorders, including alopecia and hirsutism. The objective was to investigate the influence and underlying mechanism of Krüppel-like factor 4 (KLF4) overexpression on hair follicle growth and development in C57BL/6 mice. To provide a theoretical basis for the biological functions of KLF4 gene in hair follicle development and hair follicle cycle, mice were assigned to three groups: experimental, overexpressing KLF4 (Ad-KLF4); control, expressing green fluorescent protein (Ad-NC); and blank, no treatment. Fur was removed from the dorsal surface, and the mice were intradermally injected with 25 μL 1 × 10 PFU/mL adenovirus vector (Ad-KLF4 or Ad-NC) at three points. Samples were collected for molecular biological and histological analysis. It was found that mRNA and protein levels of Wnt pathway-associated factors β-catenin, LEF1, hair follicle cell proliferation-related factor Ki67, and hair follicle inner caledrin marker AE15 were all significantly greater in the Ad-NC and blank groups than in Ad-KLF4 mice (P < 0.01). These findings were confirmed by immunohistochemical analysis. Hair growth was monitored photographically for 14 days, showing an absence of growth in the injected region of the KLF4-overexpressing mice in contrast to non-overexpressing areas where hair growth was normal. HE staining showed that hair follicles in the blank and Ad-NC mice were normal, while those in the KLF4-overexpressing areas remained in telogen or early anagen with spherical dermal papillae situated at the edge of the dermis and subcutaneous tissue without an inner heel sheath. In conclusion, it was found that KLF4 downregulated key Wnt/β-catenin-associated factors during follicular regeneration in mice, reducing both follicular development and growth.
Topics: Animals; Mice; beta Catenin; Growth and Development; Hair Follicle; Kruppel-Like Factor 4; Mice, Inbred C57BL; Wnt Proteins
PubMed: 37561178
DOI: 10.1007/s00427-023-00708-8 -
Cells Dec 2022Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher...
Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells.
Topics: Humans; Alternative Splicing; ATP-Dependent Proteases; Glycolysis; Homeostasis; Mitochondria; Mitochondrial Proteins; Neoplasms; Protein Isoforms
PubMed: 36497197
DOI: 10.3390/cells11233940 -
Open Biology Jan 2022Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during...
Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising nl1 (yeast Spc105), is12 (yeast Mtw1), and dc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.
Topics: Chromosome Segregation; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 35042402
DOI: 10.1098/rsob.210274 -
International Review of Cell and... 2020The Tyro3, Axl, and MerTK (TAM) receptors are three homologous Type I Receptor Tyrosine Kinases that have important homeostatic functions in multicellular organisms by... (Review)
Review
The Tyro3, Axl, and MerTK (TAM) receptors are three homologous Type I Receptor Tyrosine Kinases that have important homeostatic functions in multicellular organisms by regulating the clearance of apoptotic cells (efferocytosis). Pathologically, TAM receptors are overexpressed in a wide array of human cancers, and often associated with aggressive tumor grade and poor overall survival. In addition to their expression on tumor cells, TAMs are also expressed on infiltrating myeloid-derived cells in the tumor microenvironment, where they appear to act akin to negative immune checkpoints that impair host anti-tumor immunity. The ligands for TAMs are two endogenous proteins, Growth Arrest-Specific 6 (Gas6) and Protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PtdSer) on apoptotic cells and the TAM ectodomains. One interesting feature of TAMs biology is that their ligand proteins require specific post-translational modifications to acquire activities. This chapter summarized these important modifications and explained the molecular mechanisms behind such phenomenon. Current evidences suggest that these modifications help Gas6/Pros1 to achieve optimal PtdSer-binding capacities. In addition, this chapter included recent discovery of regulating machineries of PtdSer dynamic across the plasma membrane, as well as their potential impacts in the tumor microenvironment. Taken together, this review highlights the importance of the upstream PtdSer and Gas6 in regulating TAMs' function and hope to provide researchers with new perspectives to inspire future studies of TAM receptors in human disease models.
Topics: Animals; Apoptosis; Enzyme Activation; Humans; Intercellular Signaling Peptides and Proteins; Neoplasms; Protein Processing, Post-Translational; Protein S; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Tumor Microenvironment; c-Mer Tyrosine Kinase; Axl Receptor Tyrosine Kinase
PubMed: 33234244
DOI: 10.1016/bs.ircmb.2020.09.002 -
Cells Apr 2023Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its...
Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway.
Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its efficacy is severely limited by cell aging and senescence. Gremlin-1 (GREM1), a member of the bone morphogenetic protein antagonist family, has been implicated in cell proliferation and survival. However, GREM1's role in cell aging and senescence has never been investigated in human cardiac mesenchymal progenitor cells (hMPCs). Therefore, this study assessed the hypothesis that overexpression of GREM1 rejuvenates the cardiac regenerative potential of aging hMPCs to a youthful stage and therefore allows better capacity for myocardial repair. We recently reported that a subpopulation of hMPCs with low mitochondrial membrane potential can be sorted from right atrial appendage-derived cells in patients with cardiomyopathy and exhibit cardiac reparative capacity in a mouse model of myocardial infarction. In this study, lentiviral particles were used to overexpress GREM1 in these hMPCs. Protein and mRNA expression were assessed through Western blot and RT-qPCR. FACS analysis for Annexin V/PI staining and lactate dehydrogenase assay were used to assess cell survival. It was observed that cell aging and cell senescence led to a decrease in GREM1 expression. In addition, overexpression of GREM1 led to a decrease in expression of senescence genes. Overexpression of GREM1 led to no significant change in cell proliferation. However, GREM1 appeared to have an anti-apoptotic effect, with an increase in survival and decrease in cytotoxicity evident in GREM1-overexpressing hMPCs. Overexpressing GREM1 also induced cytoprotective properties by decreasing reactive oxidative species and mitochondrial membrane potential. This result was associated with increased expression of antioxidant proteins, such as SOD1 and catalase, and activation of the ERK/NRF2 survival signal pathway. Inhibition of ERK led to a decrease in GREM1-mediated rejuvenation in terms of cell survival, which suggests that an ERK-dependent pathway may be involved. Taken altogether, these results indicate that overexpression of GREM1 can allow aging hMPCs to adopt a more robust phenotype with improved survival capacity, which is associated with an activated ERK/NRF2 antioxidant signal pathway.
Topics: Animals; Mice; Humans; Aged; Antioxidants; Up-Regulation; NF-E2-Related Factor 2; Signal Transduction; Mesenchymal Stem Cells; Bone Morphogenetic Proteins; Intercellular Signaling Peptides and Proteins
PubMed: 37190112
DOI: 10.3390/cells12081203 -
The Journal of Investigative Dermatology Dec 2023Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of cancer with metastatic potential. MicroRNAs regulate gene expression at the...
Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of cancer with metastatic potential. MicroRNAs regulate gene expression at the post-transcriptional level. In this study, we report that miR-23b is downregulated in cSCCs and in actinic keratosis and that its expression is regulated by the MAPK signaling pathway. We show that miR-23b suppresses the expression of a gene network associated with key oncogenic pathways and that the miR-23b-gene signature is enriched in human cSCCs. miR-23b decreased the expression of FGF2 both at mRNA and protein levels and impaired the angiogenesis-inducing ability of cSCC cells. miR23b overexpression suppressed the capacity of cSCC cells to form colonies and spheroids, whereas the CRISPR/Cas9-mediated deletion of MIR23B resulted in increased colony and tumor sphere formation in vitro. In accordance with this, miR-23b-overexpressing cSCC cells formed significantly smaller tumors upon injection into immunocompromised mice with decreased cell proliferation and angiogenesis. Mechanistically, we verify RRAS2 as a direct target of miR-23b in cSCC. We show that RRAS2 is overexpressed in cSCC and that interference with its expression impairs angiogenesis and colony and tumorsphere formation. Taken together, our results suggest that miR-23b acts in a tumor-suppressive manner in cSCC, and its expression is decreased during squamous carcinogenesis.
Topics: Humans; Animals; Mice; Carcinoma, Squamous Cell; Skin Neoplasms; Signal Transduction; Carcinogenesis; MicroRNAs; Membrane Proteins; Monomeric GTP-Binding Proteins
PubMed: 37423552
DOI: 10.1016/j.jid.2023.05.026