-
International Journal of Molecular... Nov 2023The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with...
The function of the α-adrenergic receptor phosphorylation sites previously detected by mass spectrometry was evaluated by employing mutants, substituting them with non-phosphorylatable amino acids. Substitution of the intracellular loop 3 (IL3) sites did not alter baseline or stimulated receptor phosphorylation, whereas substitution of phosphorylation sites in the carboxyl terminus (Ctail) or both domains (IL3/Ctail) markedly decreased receptor phosphorylation. Cells expressing the IL3 or Ctail receptor mutants exhibited a noradrenaline-induced calcium-maximal response similar to those expressing the wild-type receptor, and a shift to the left in the concentration-response curve to noradrenaline was also noticed. Cells expressing the IL3/Ctail mutant exhibited higher apparent potency and increased maximal response to noradrenaline than those expressing the wild-type receptor. Phorbol ester-induced desensitization of the calcium response to noradrenaline was reduced in cells expressing the IL3 mutant and abolished in cells in which the Ctail or the IL3/Ctail were modified. In contrast, desensitization in response to preincubation with noradrenaline was unaffected in cells expressing the distinct receptor mutants. Noradrenaline-induced ERK phosphorylation was surprisingly increased in cells expressing IL3-modified receptors but not in those expressing receptors with the Ctail or IL3/Ctail substitutions. Our data indicate that phosphorylation sites in the IL3 and Ctail domains mediate and regulate α-adrenergic receptor function. Phorbol ester-induced desensitization seems to be closely associated with receptor phosphorylation, whereas noradrenaline-induced desensitization likely involves other elements.
Topics: Phosphorylation; Calcium; Norepinephrine; Phorbol Esters; Receptors, Adrenergic
PubMed: 38069285
DOI: 10.3390/ijms242316963 -
Bioorganic & Medicinal Chemistry Letters Jan 2024Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well...
Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well established anti-cancer activity, most natural products that target the PKC regulator domain exhibit tumor promotion properties. In this study, we examine a focused library of indolactam analogues in cell-based assays to establish the structural features of the scaffold that enhance bryostatin 1-like activity. These systematic biological assessments identified specific indole substitution patterns that impart diminished tumor promotion behavior in vitro for indolactam analogues, while still maintaining nanomolar potency for PKC.
Topics: Humans; Bryostatins; Lactones; Neoplasms; Protein Kinase C; Tetradecanoylphorbol Acetate; Lactams
PubMed: 38036273
DOI: 10.1016/j.bmcl.2023.129570 -
Anais Da Academia Brasileira de Ciencias 2023Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most...
Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most prevalent. Although the current therapy is often effective, it is associated with adverse effects and the possibility of drug tolerance. N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline is a L-proline amino acid derivative found in the leaves of Sideroxylon obtusifolium, a species traditionally used to treat inflammatory diseases. The aim of this study was to investigate the topical anti-inflammatory effect of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis in mice. Topically administered NMP, at doses of 0.03 - 0.50 mg/ear, reduced TPA-induced ear edema and neutrophil migration, as evidenced by low tissue myeloperoxidase activity and verified by histological examination. In addition, NMP (0.06 mg/ear) reduced tissue levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, INF-γ and MCP-1) and of the anti-inflammatory cytokine IL-10, and reduced gene expression of TNF-α, IL-6 and IL-1β increased by TPA. The data suggest that N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline acts as a topical anti-inflammatory agent that decreases the expression of inflammatory cytokines, making it useful for the treatment of skin inflammation. Further investigations are necessary for its development as a therapeutic agent.
Topics: Mice; Animals; Tetradecanoylphorbol Acetate; Irritants; Tumor Necrosis Factor-alpha; Interleukin-6; Dermatitis, Contact; Anti-Inflammatory Agents; Dermatitis; Cytokines; Sapotaceae
PubMed: 37909544
DOI: 10.1590/0001-3765202320220919 -
ELife Oct 2023Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt...
Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here, we show that a macropinocytosis activator, the tumor promoter phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.
Topics: Female; Pregnancy; Humans; Animals; Mice; Carcinogens; Wnt Signaling Pathway; Glycogen Synthase Kinase 3; Phorbol Esters; Esters; Neoplasms
PubMed: 37902809
DOI: 10.7554/eLife.89141 -
Zhong Nan Da Xue Xue Bao. Yi Xue Ban =... Aug 2023The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy....
OBJECTIVES
The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.
METHODS
The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO group, a LY294002 group, a SC79 group, a LY294002+SiO group, and a SC79+SiO group were set up in this experiment. Macrophages in the LY294002+SiO group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting.
RESULTS
After the macrophages were exposed to SiO dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all <0.05). When 100 μg/mL SiO dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all <0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all <0.05). Immunofluorescence results demonstrated that after exposure to SiO (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO group (all <0.05). Compared with the SiO group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all <0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both <0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all <0.05) in the LY294002+SiO group. Compared with the SiO group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all <0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both <0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all <0.05) in the SC79+SiO group.
CONCLUSIONS
Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.
Topics: Humans; Proto-Oncogene Proteins c-akt; Transforming Growth Factor beta1; Silicon Dioxide; Phosphatidylinositol 3-Kinases; Matrix Metalloproteinase 1; Tissue Inhibitor of Metalloproteinase-1; Sirolimus; Beclin-1; Tumor Necrosis Factor-alpha; Dust; TOR Serine-Threonine Kinases; Lung; Fibroblasts; Silicosis; Macrophages; Autophagy
PubMed: 37875355
DOI: 10.11817/j.issn.1672-7347.2023.220581 -
BioRxiv : the Preprint Server For... Oct 2023Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their...
Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.
PubMed: 37873256
DOI: 10.1101/2023.10.11.561954 -
Toxicology Letters Nov 2023To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been...
To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha-ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12-O-tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents.
Topics: Animals; Mice; Transcriptome; Butylated Hydroxyanisole; Reproducibility of Results; Quercetin; Carcinogenicity Tests; BALB 3T3 Cells; Cell Transformation, Neoplastic; Carcinogens; Tetradecanoylphorbol Acetate; Cholic Acid
PubMed: 37813191
DOI: 10.1016/j.toxlet.2023.10.006 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... Sep 2023Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According... (Review)
Review
Tigliane type macrocyclic diterpenoids with special structures and diverse bioactivities are mainly extracted from plants of Euphorbiaceae and Thymelaeaceae. According to the different functional groups, they can be classified into types of phorbol esters, C-4 deoxyphorbol esters, C-12 deoxyphorbol esters, C-16 or C-17 substituted phorbol esters and others. Most of them present promising antiviral activities and cytotoxic activities and are expected to be developed as candidates for anti-AIDS, anti-tuberculosis, and anti-tumor clinical trials, demonstrating great potential for the application in healthcare. This paper reviews 115 novel tigliane-type diterpenoids discovered since 2013 and summarize their chemical structures and bioactivities, aiming to lay a foundation for further development and utilization of these compounds and provide new ideas for the development of clinical drugs.
Topics: Phorbols; Molecular Structure; Diterpenes; Antiviral Agents; Phorbol Esters
PubMed: 37802801
DOI: 10.19540/j.cnki.cjcmm.20230427.201 -
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 -
Scientific Reports Sep 2023Stimulation of glucose uptake in response to ischemic metabolic stress is important for cardiomyocyte function and survival. Chronic exposure of cardiomyocytes to fatty...
Stimulation of glucose uptake in response to ischemic metabolic stress is important for cardiomyocyte function and survival. Chronic exposure of cardiomyocytes to fatty acids (FA) impairs the stimulation of glucose uptake, whereas induction of lipid droplets (LD) is associated with preserved glucose uptake. However, the mechanisms by which LD induction prevents glucose uptake impairment remain elusive. We induced LD with either tetradecanoyl phorbol acetate (TPA) or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Triacylglycerol biosynthesis enzymes were inhibited in cardiomyocytes exposed to FA ± LD inducers, either upstream (glycerol-3-phosphate acyltransferases; GPAT) or downstream (diacylglycerol acyltransferases; DGAT) of the diacylglycerol step. Although both inhibitions reduced LD formation in cardiomyocytes treated with FA and LD inducers, only DGAT inhibition impaired metabolic stress-stimulated glucose uptake. DGAT inhibition in FA plus TPA-treated cardiomyocytes reduced triacylglycerol but not diacylglycerol content, thus increasing the diacylglycerol/triacylglycerol ratio. In cardiomyocytes exposed to FA alone, GPAT inhibition reduced diacylglycerol but not triacylglycerol, thus decreasing the diacylglycerol/triacylglycerol ratio, prevented PKCδ activation and improved metabolic stress-stimulated glucose uptake. Changes in AMP-activated Protein Kinase activity failed to explain variations in metabolic stress-stimulated glucose uptake. Thus, LD formation regulates metabolic stress-stimulated glucose uptake in a manner best reflected by the diacylglycerol/triacylglycerol ratio.
Topics: Myocytes, Cardiac; Biological Transport; Diacylglycerol O-Acyltransferase; Fatty Acids; Tetradecanoylphorbol Acetate; Glucose
PubMed: 37684349
DOI: 10.1038/s41598-023-42072-7