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Medicina (Kaunas, Lithuania) Jun 2024: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in...
: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. : Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. : Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11b THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DR and CD206), M1 (HLA-DR and CD206), and M2 (HLA-DR and CD206), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. : We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.
Topics: Humans; Macrophages; Tetradecanoylphorbol Acetate; Flavonoids; THP-1 Cells; Cell Differentiation; Monocytes; Flow Cytometry; Phagocytosis
PubMed: 38929626
DOI: 10.3390/medicina60061009 -
International Journal of Molecular... Jun 2024LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased...
LPA receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin-LPA receptor association. The agonist and the phorbol ester-induced marked LPA receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA receptor signaling and regulation.
Topics: Humans; Receptors, Lysophosphatidic Acid; Phosphorylation; HEK293 Cells; Signal Transduction; Lysophospholipids; beta-Arrestins; Binding Sites; Calcium Signaling
PubMed: 38928196
DOI: 10.3390/ijms25126491 -
Biomolecules May 2024Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and...
Nuclear hormone receptors exist in dynamic equilibrium between transcriptionally active and inactive complexes dependent on interactions with ligands, proteins, and chromatin. The present studies examined the hypothesis that endogenous ligands activate peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in keratinocytes. The phorbol ester treatment or HRAS infection of primary keratinocytes increased fatty acids that were associated with enhanced PPARβ/δ activity. Fatty acids caused PPARβ/δ-dependent increases in chromatin occupancy and the expression of angiopoietin-like protein 4 () mRNA. Analyses demonstrated that stearoyl Co-A desaturase 1 () mediates an increase in intracellular monounsaturated fatty acids in keratinocytes that act as PPARβ/δ ligands. The activation of PPARβ/δ with palmitoleic or oleic acid causes arrest at the G2/M phase of the cell cycle of HRAS-expressing keratinocytes that is not found in similarly treated HRAS-expressing -null keratinocytes. HRAS-expressing -null mouse keratinocytes exhibit enhanced cell proliferation, an effect that is mitigated by treatment with palmitoleic or oleic acid. Consistent with these findings, the ligand activation of PPARβ/δ with GW0742 or oleic acid prevented UVB-induced non-melanoma skin carcinogenesis, an effect that required PPARβ/δ. The results from these studies demonstrate that PPARβ/δ has endogenous roles in keratinocytes and can be activated by lipids found in diet and cellular components.
Topics: Keratinocytes; PPAR-beta; Animals; Mice; Stearoyl-CoA Desaturase; PPAR delta; Fatty Acids; Angiopoietin-Like Protein 4; Humans; Oleic Acid; Proto-Oncogene Proteins p21(ras); Fatty Acids, Monounsaturated; Skin Neoplasms
PubMed: 38927010
DOI: 10.3390/biom14060606 -
Ecotoxicology and Environmental Safety Jul 2024The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA...
The correlation between formaldehyde (FA) exposure and prevalence of asthma has been widely reported. However, the underlying mechanism is still not fully understood. FA exposure at 2.0 mg/m was found to exacerbate asthma in OVA-induced murine models. IFN-γ, the cytokine produced by T helper 1 (Th1) cells, was significantly induced by FA in serum and bronchoalveolar lavage fluid (BALF) of asthmatic mice, which was different from cytokines secreted by other Th cells. The observation was also confirmed by mRNA levels of Th marker genes in CD4+ T cells isolated from BALF. In addition, increased production of IFN-γ and expression of T-bet in Jurkat T cells primed with phorbol ester and phytohaemagglutinin were also observed with 100 μM FA treatment in vitro. Upregulated STAT1 phosphorylation, T-bet expression and IFN-γ production induced by FA was found to be restrained by STAT1 inhibitor fludarabine, indicating that FA promoted Th1 commitment through the autocrine IFN-γ/STAT1/T-bet pathway in asthma. This work not only revealed that FA could bias Th lineage commitment to exacerbate allergic asthma, but also identified the signaling mechanism of FA-induced Th1 differentiation, which may be utilized as the target for development of interfering strategies against FA-induced immune disorders.
Topics: Asthma; Animals; STAT1 Transcription Factor; Interferon-gamma; Mice; T-Box Domain Proteins; Formaldehyde; Inflammation; Mice, Inbred BALB C; Humans; Female; Bronchoalveolar Lavage Fluid; T-Lymphocytes, Helper-Inducer; Signal Transduction; Th1 Cells; Jurkat Cells
PubMed: 38823345
DOI: 10.1016/j.ecoenv.2024.116534 -
Microorganisms Apr 2024Chagas disease is caused by the single-flagellated protozoan , which affects several million people worldwide. Understanding the signal transduction pathways involved in...
In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Epimastigotes.
Chagas disease is caused by the single-flagellated protozoan , which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in epimastigotes.
PubMed: 38792752
DOI: 10.3390/microorganisms12050907 -
Pharmaceuticals (Basel, Switzerland) Mar 2024Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca concentration ([Ca]) in pulmonary artery...
Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca concentration ([Ca]) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca] through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O/5% CO for 24 h to elucidate TRPC1 overexpression and observe the Ca release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca entry upon SR Ca depletion in hypoxic PASMCs.
PubMed: 38675401
DOI: 10.3390/ph17040440 -
International Journal of Molecular... Apr 2024Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under...
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.
Topics: Humans; c-Mer Tyrosine Kinase; ADAM17 Protein; Amyloid Precursor Protein Secretases; Proteolysis; Intercellular Signaling Peptides and Proteins; THP-1 Cells; Macrophages; Protein S; Monocytes; Tetradecanoylphorbol Acetate
PubMed: 38673989
DOI: 10.3390/ijms25084404 -
Biomolecules & Therapeutics May 2024In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on...
In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.
PubMed: 38589300
DOI: 10.4062/biomolther.2023.186 -
Biochimica Et Biophysica Acta.... Jun 2024Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of...
Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.
Topics: Humans; Cytochromes c; Megakaryocytes; Mitochondria; K562 Cells; Thrombocytopenia; Apoptosis; Thrombopoiesis; Mutation
PubMed: 38531481
DOI: 10.1016/j.bbadis.2024.167134 -
NPJ Systems Biology and Applications Mar 2024Skin cancer and other skin-related inflammatory pathologies are rising due to heightened exposure to environmental pollutants and carcinogens. In this context, natural...
Skin cancer and other skin-related inflammatory pathologies are rising due to heightened exposure to environmental pollutants and carcinogens. In this context, natural products and repurposed compounds hold promise as novel therapeutic and preventive agents. Strengthening the skin's antioxidant defense mechanisms is pivotal in neutralizing reactive oxygen species (ROS) and mitigating oxidative stress. Sunset Yellow (SY) exhibits immunomodulatory characteristics, evidenced by its capacity to partially inhibit the secretion of proinflammatory cytokines, regulate immune cell populations, and modulate the activation of lymphocytes. This study aimed to investigate the antioxidant and anti-genotoxic properties of SY using in-silico, in vitro, and physiochemical test systems, and to further explore its potential role in 7,12-dimethylbenz(a) anthracene (DMBA)/ 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage skin carcinogenesis. In vitro experiments showed that pre-treatment of SY significantly enhanced the cell viability of HaCaT cells when exposed to tertiary-Butyl Hydrogen Peroxide (tBHP). This increase was accompanied by reduced ROS levels, restoration of mitochondrial membrane potential, and notable reduction in DNA damage in (SY + tBHP) treated cells. Mechanistic investigations using DPPH chemical antioxidant activity test and potentiometric titrations confirmed SY's antioxidant properties, with a standard reduction potential ( ) of 0.211 V. Remarkably, evaluating the effect of topical application of SY in DMBA/TPA-induced two-step skin carcinogenesis model revealed dose-dependent decreases in tumor latency, incidence, yield, and burden over 21-weeks. Furthermore, computational analysis and experimental validations identified GSK3β, KEAP1 and EGFR as putative molecular targets of SY. Collectively, our findings reveal that SY enhances cellular antioxidant defenses, exhibits anti-genotoxic effects, and functions as a promising chemopreventive agent.
Topics: Humans; Kelch-Like ECH-Associated Protein 1; Antioxidants; Reactive Oxygen Species; NF-E2-Related Factor 2; 9,10-Dimethyl-1,2-benzanthracene; Skin Neoplasms; Tetradecanoylphorbol Acetate; Oxidative Stress; Chemoprevention; Carcinogenesis; Azo Compounds
PubMed: 38431714
DOI: 10.1038/s41540-024-00349-1