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Molecular Biology Reports Nov 2020Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum...
Numerous protocols to establish dopaminergic phenotype in SH-SY5Y cells have been reported. In most of these protocols there are variations in concentration of serum used. In this paper, we compared the effects of high (10%), low (3%) and descending (2.5%/1%) serum concentration in differentiation medium containing different proportion of retinoic acid (RA) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) or RA-only on the undifferentiated SH-SY5Y cells with regards to cell morphology, biochemical and gene expression alterations. Cells differentiated in culture medium containing low and descending serum concentrations showed increased number of neurite projections and reduced proliferation rates when compared to undifferentiated cells. The SH-SY5Y cells differentiated in culture medium containing 3% RA and low serum or descending (2.5%/1% RA/TPA) were found to be more susceptible to 6-hydroxydopamine (6-OHDA) induced cytotoxicity. Cells differentiated with RA/TPA or RA differentiated showed increased production of the α-synuclein (SNCA) neuroprotein and dopamine neurotransmitter compared to undifferentiated cells, regardless serum concentrations used. There was no significant difference in the expression of tyrosine hydroxylase (TH) gene between undifferentiated and differentiated SH-SY5Y cells. However, the expression of dopamine receptor D2 (DRD2) gene was markedly increased (p<0.05) in differentiated cells with 3% serum and RA only when compared to undifferentiated cells. In conclusion, to terminally differentiate SH-SY5Y cells to be used as a cell-based model to study Parkinson's disease (PD) to investigate molecular mechanisms and drug discovery, the optimal differentiation medium should contain 3% serum in RA-only.
Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Culture Media; Gene Expression Regulation; Humans; Phorbol Esters; Receptors, Dopamine D2; Tretinoin; Tyrosine 3-Monooxygenase; alpha-Synuclein
PubMed: 33098048
DOI: 10.1007/s11033-020-05925-2 -
PKCβII activation requires nuclear trafficking for phosphorylation and Mdm2-mediated ubiquitination.Life Science Alliance Apr 2023PKCβII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and...
PKCβII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and mutants of PKCβII with altered phosphorylation and protein interaction behaviors to identify the cellular mechanisms underlying the activation of PKCβII. Our results show that 3-phosphoinositide-dependent protein kinase-1 (PDK1)-mediated constitutive phosphorylation of PKCβII at the activation loop (T500) is required for phorbol ester-induced nuclear entry and subsequent Mdm2-mediated ubiquitination of PKCβII, whereas ubiquitination of PKCβII is required for the PDK1-mediated inducible phosphorylation of PKCβII at T500 in the nucleus. After moving out of the nucleus, PKCβII interacts with actin, undergoes inducible mTORC2-mediated phosphorylation at the turn motif (T641), interacts with clathrin, and then translocates to the plasma membrane. This overall cascade of cellular events intertwined with the phosphorylation at critical residues and Mdm2-mediated ubiquitination in the nucleus and along with interactions with actin and clathrin plays roles that encompass the core processes of PKC activation.
Topics: Actins; Clathrin; Phosphorylation; Protein Kinase C beta; Ubiquitination; Proto-Oncogene Proteins c-mdm2
PubMed: 36717249
DOI: 10.26508/lsa.202201748 -
Carcinogenesis Nov 2019XB130 is an adaptor protein that functions as a mediator of multiple tyrosine kinases important for regulating cell proliferation, survival, migration and invasion....
XB130 is an adaptor protein that functions as a mediator of multiple tyrosine kinases important for regulating cell proliferation, survival, migration and invasion. Formerly predicted as an oncogene, alterations of its expression are documented in various human cancers. However, the exact role of XB130 in tumorigenesis is unknown. To address its function in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on XB130 knockout (KO), heterozygous (HZ) and wild-type (WT) littermate mice. DMBA/TPA-treated XB130 KO and HZ males developed a significantly higher number of epidermal tumors that were notably larger in size than did WT mice. Interestingly, DMBA/TPA-treated female mice did not show any difference in tumor multiplicity regardless of the genotypes. The skin tumor lesions of XB130 KO males were more progressed with an increased frequency of keratoacanthoma. Deficiency of XB130 dramatically increased epidermal tumor cell proliferation. The responses to DMBA and TPA stimuli were also individually investigated to elucidate the mechanistic role of XB130 at different stages of tumorigenesis. DMBA-treated male XB130 KO mice showed compensatory p53-mediated stress response. TPA-treated XB130 KO males demonstrated more skin ulceration with more severe edema, enhanced cell proliferation, accumulation of infiltrating neutrophils and increased production of pro-inflammatory cytokine genes compared with WT mice. Enhanced activities of nuclear factor-kappa B pathway, increased protein expression of metalloproteinase-9 and ERK1/2 phosphorylation were found in these KO mice. These findings demonstrate that XB130 acts as a tumor suppressor in carcinogen-induced skin tumorigenesis that may be mediated through inhibiting inflammation.
Topics: 9,10-Dimethyl-1,2-benzanthracene; Adaptor Proteins, Signal Transducing; Animals; Carcinogenesis; Carcinogens; Cell Proliferation; Female; Genes, Tumor Suppressor; Inflammation; Male; Mice; Mice, Knockout; Microfilament Proteins; Skin Neoplasms; Tetradecanoylphorbol Acetate
PubMed: 30820526
DOI: 10.1093/carcin/bgz042 -
Free Radical Research Dec 2019Matrix metalloproteinases (MMPs), zinc-containing proteinases, play a critical role in tumour progression by degrading extracellular matrix components. MMP2 and MMP9 are...
Matrix metalloproteinases (MMPs), zinc-containing proteinases, play a critical role in tumour progression by degrading extracellular matrix components. MMP2 and MMP9 are secreted from tumour-associated macrophages as well as tumour cells and have been implicated in the formation of the tumour microenvironment. Therefore, the inhibition of these MMPs may suppress tumour progression and metastasis. 4-Hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE) is known to cause apoptosis in the human lung cancer cell line A549 by inducing endoplasmic reticulum (ER) stress. However, the effects of HPO-DAEE on tumour progression remain unclear. HPO-DAEE pre-treatment significantly suppressed phorbol 12-myristate 13-acetate (TPA)-triggered MMP activation in human monocytic THP-1 cells. It also enhanced the expression of haem oxygenase-1, an antioxidant enzyme, and suppressed the TPA-triggered intracellular accumulation of reactive oxygen species (ROS). Furthermore, HPO-DAEE suppressed transforming growth factor-β1-triggered human prostate cancer PC3 cell migration and this was accompanied by the inhibition of MMP expression and activities. The present results indicate that HPO-DAEE may exert inhibitory effects on tumour progression by suppressing MMP expression and activities.
Topics: Antineoplastic Agents; Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Esters; Fatty Acids; Fatty Acids, Monounsaturated; Humans; Male; Matrix Metalloproteinases; Phorbol Esters; Prostatic Neoplasms; Reactive Oxygen Species; Structure-Activity Relationship; Transforming Growth Factor beta1
PubMed: 31575304
DOI: 10.1080/10715762.2019.1675874 -
Anti-cancer Agents in Medicinal... 2021Medicinal plants serve as sources of compounds used to treat other types of cancers. The root of the plant Lophira alata (Ochnaceae) has been used as a component of...
BACKGROUND
Medicinal plants serve as sources of compounds used to treat other types of cancers. The root of the plant Lophira alata (Ochnaceae) has been used as a component of traditional herbal decoctions administered to cancer patients in southwestern Nigeria. However, the mechanism of the cytotoxic effects of Lophira alata alone or in the presence of phorbol ester has not been investigated in brain tumor cells.
OBJECTIVE
This study aimed to examine the cytotoxic potential of the methanolic fraction of Lophira alata root on malignant glioma invasive cellular growth and survival.
METHODS
The methanolic fraction of Lophira alata (LAM) was subjected to high-performance liquid chromatography to determine the fingerprints of the active molecules. The antiproliferative effects of Lophira alata were assessed using the MTT and LDH assays. Protein immunoblots were carried out to test the effects of Lophira alata, alone or in the presence of phorbol ester, on survival signaling pathways, such as Akt, mTOR, and apoptotic markers such as PARP and caspases.
RESULTS
The methanolic fraction of Lophira alata (LAM) induced a concentration-dependent and time-dependent decrease in glioma cell proliferation. In addition, LAM attenuated phorbol ester-mediated signaling of downstream targets such as Akt/mTOR. Gene silencing using siRNA targeting PKC-alpha attenuated LAM-mediated downregulation of Akt. In addition, LAM induced both PARP and caspase cleavages. The HPLC fingerprint of the fraction indicates the presence of flavonoids.
CONCLUSION
LAM decreases cell proliferation and induces apoptosis in glioma cell lines and thus could serve as a therapeutic molecule in the management of gliomas.
Topics: Antineoplastic Agents, Phytogenic; Cell Proliferation; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Ochnaceae; Phorbol Esters; Plant Extracts; Protein Kinase C-alpha; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Tumor Cells, Cultured
PubMed: 33622229
DOI: 10.2174/1871520621666210223094235 -
BMC Biology Sep 2021Maturation is a process that allows synapses to acquire full functionality, optimizing their activity to diverse neural circuits, and defects in synaptic maturation may...
BACKGROUND
Maturation is a process that allows synapses to acquire full functionality, optimizing their activity to diverse neural circuits, and defects in synaptic maturation may contribute to neurodevelopmental disorders. Neuroligin-1 (NL1) is a postsynaptic cell adhesion molecule essential for synapse maturation, a role typically attributed to binding to pre-synaptic ligands, the neurexins. However, the pathways underlying the action of NL1 in synaptic maturation are incompletely understood, and some of its previously observed effects seem reminiscent of those described for the neurotrophin brain-derived neurotrophic factor (BDNF). Here, we show that maturational increases in active zone stability and synaptic vesicle recycling rely on the joint action of NL1 and brain-derived neurotrophic factor (BDNF).
RESULTS
Applying BDNF to hippocampal neurons in primary cultures or organotypical slice cultures mimicked the effects of overexpressing NL1 on both structural and functional maturation. Overexpressing a NL1 mutant deficient in neurexin binding still induced presynaptic maturation. Like NL1, BDNF increased synaptic vesicle recycling and the augmentation of transmitter release by phorbol esters, both hallmarks of presynaptic maturation. Mimicking the effects of NL1, BDNF also increased the half-life of the active zone marker bassoon at synapses, reflecting increased active zone stability. Overexpressing NL1 increased the expression and synaptic accumulation of BDNF. Inhibiting BDNF signaling pharmacologically or genetically prevented the effects of NL1 on presynaptic maturation. Applying BDNF to NL1-knockout mouse cultures rescued defective presynaptic maturation, indicating that BDNF acts downstream of NL1 and can restore presynaptic maturation at late stages of network development.
CONCLUSIONS
Our data introduce BDNF as a novel and essential component in a transsynaptic pathway linking NL1-mediated cell adhesion, neurotrophin action, and presynaptic maturation. Our findings connect synaptic cell adhesion and neurotrophin signaling and may provide a therapeutic approach to neurodevelopmental disorders by targeting synapse maturation.
Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Adhesion Molecules, Neuronal; Cells, Cultured; Hippocampus; Mice; Mice, Knockout; Neurons; Signal Transduction; Synapses
PubMed: 34579720
DOI: 10.1186/s12915-021-01145-7 -
Frontiers in Immunology 2022Although recent studies have highlighted the link of TIPE2 and asthma airway inflammation, its roles and molecular mechanisms in different asthma inflammatory phenotypes...
PURPOSE
Although recent studies have highlighted the link of TIPE2 and asthma airway inflammation, its roles and molecular mechanisms in different asthma inflammatory phenotypes remain largely unknown. We evaluated sputum TIPE2 expression level and its correlation with different asthma phenotypes. Additionally, we explored the roles and mechanism of TIPE2 in M1 polarization of macrophages.
METHODS
A total of 102 asthma patients who underwent sputum induction were enrolled to evaluate the expression level of TIPE2 and its association with different asthma phenotypes. To explore the roles and mechanism of TIPE2 in M1 polarization of macrophages, THP-1 monocytes stimulated with phorbol-12-myristate-13-acetate, were used as a model of undifferentiated (M0) macrophages, and M0 macrophages were treated with lipopolysaccharide to induce M1 macrophages.
RESULTS
The sputum TIPE2 level was significantly lower in patients with neutrophilic asthma (NA) and higher in patients with eosinophilic asthma (EA) compared with patients with paucigranulocytic asthma. The levels of IL-1β, TNF-α and IL-6 were highest in NA compared with other groups. TIPE2 levels in sputum negatively correlated with IL-1β and TNF-α levels but positively correlated with IL-4, IL-5, IL-13, and IL-10 levels ( < 0.05). , TIPE2 enhanced Nrf2/HO-1 pathway activation in macrophages and inhibited LPS-induced M1 macrophage differentiation and related cytokine release. Further analysis showed that the Nrf2 inhibitor ML385 weakened TIPE2-induced activation of the Nrf2/HO-1 pathway, as well as TIPE2-induced suppression in M1 polarization of macrophage and inflammatory cytokines secretion.
CONCLUSIONS
TIPE2 expression level was highly down-regulated in NA and was negatively correlated with inflammatory factors (IL-1β and TNF-α). Aberrant expression of TIPE2 may target the Nrf2/HO-1 pathway to inhibit M1 macrophage-related neutrophilic inflammation in asthma.
Topics: Asthma; Heme Oxygenase-1; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Lipopolysaccharides; Macrophages; NF-E2-Related Factor 2; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35572500
DOI: 10.3389/fimmu.2022.883885 -
Pflugers Archiv : European Journal of... Jan 2024Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal...
Particularly expressed in the kidney, αKlotho is a transmembrane protein that acts together with bone hormone fibroblast growth factor 23 (FGF23) to regulate renal phosphate and vitamin D homeostasis. Soluble Klotho (sKL) is released from the transmembrane form and controls various cellular functions as a paracrine and endocrine factor. αKlotho deficiency accelerates aging, whereas its overexpression favors longevity. Higher αKlotho abundance confers a better prognosis in cardiovascular and renal disease owing to anti-inflammatory, antifibrotic, or antioxidant effects and tumor suppression. Serine/threonine protein kinase C (PKC) is ubiquitously expressed, affects several cellular responses, and is also implicated in heart or kidney disease as well as cancer. We explored whether PKC is a regulator of αKlotho. Experiments were performed in renal MDCK or NRK-52E cells and PKC isoform and αKlotho expression determined by qRT-PCR and Western Blotting. In both cell lines, PKC activation with phorbol ester phorbol-12-myristate-13-acetate (PMA) downregulated, while PKC inhibitor staurosporine enhanced αKlotho mRNA abundance. Further experiments with PKC inhibitor Gö6976 and RNA interference suggested that PKCγ is the major isoform for the regulation of αKlotho gene expression in the two cell lines. In conclusion, PKC is a negative regulator of αKlotho gene expression, an effect which may be relevant for the unfavorable effect of PKC on heart or kidney disease and tumorigenesis.
Topics: Humans; Protein Kinase C; Glucuronidase; Fibroblast Growth Factors; Protein Isoforms; Gene Expression; Kidney Diseases
PubMed: 37773536
DOI: 10.1007/s00424-023-02863-3 -
Cell Cycle (Georgetown, Tex.) Mar 2022Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell...
Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.
Topics: Animals; Dense Core Vesicles; Exocytosis; PC12 Cells; Phorbol Esters; Rats; Synaptic Vesicles
PubMed: 35067177
DOI: 10.1080/15384101.2022.2026576 -
Journal of Cell Science Feb 2021KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However,...
KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.
Topics: Active Transport, Cell Nucleus; HeLa Cells; Humans; KRIT1 Protein; Phosphorylation; Protein Kinase C-alpha; Tetradecanoylphorbol Acetate
PubMed: 33443102
DOI: 10.1242/jcs.250217