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Molecular & Cellular Proteomics : MCP Jun 2019Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide...
Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.
Topics: Antioxidants; Collagen; Dermis; Dithiothreitol; Down-Regulation; Endoplasmic Reticulum Stress; Extracellular Matrix Proteins; Fibroblasts; Homeostasis; Humans; Mitogen-Activated Protein Kinases; Oxidation-Reduction; Phosphorylation; Phosphorylcholine; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 30890563
DOI: 10.1074/mcp.RA118.001140 -
Oncotarget Sep 2017Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains...
Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains unclear. Herein, we demonstrated that compared with T4 treatment alone, pretreatment with wortmannin (an inhibitor of phosphatidylinositol 3-kinase), perifosine (an inhibitor of AKT) or rapamycin (an inhibitor of mTOR) combined with a subsequent T4 treatment significantly impaired the cell viability of A549 and A549/DDP lung cancer cells. We found that either treatment scheme markedly reduced the activity of P13K and AKT. Expression of LC3II increased in parallel to the increase of the T4 concentration in both A549 and A549/DDP cells and was repressed by overexpression of AKT. The expression levels of PI3-K, PI3-P, AKT, TSC2, mTOR, p70S6K and 4E-BP1 were minimally affected by the wortmannin, perifosine, or rapamycin plus T4 treatments, but their phosphorylated products were greatly affected in A549 lung cancer cells and slightly affected in A549/DDP lung cancer cells. These results indicate that T4 induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway. We further found that T4 decreased expression of MDR1 and improved cisplatin sensitivity of A549/DDP cells. Altogether, these results have meaningful implications for tumor therapy in the future.
PubMed: 28969040
DOI: 10.18632/oncotarget.19201 -
PloS One 2015Although high density lipoprotein (HDL) improves the functions of endothelial progenitor cells (EPCs), the effect of HDL ApoAI mimetic peptide reverse-D-4F (Rev-D4F) on...
Although high density lipoprotein (HDL) improves the functions of endothelial progenitor cells (EPCs), the effect of HDL ApoAI mimetic peptide reverse-D-4F (Rev-D4F) on EPC mobilization and repair of EPC dysfunctions remains to be studied. In this study, we investigated the effects of Rev-D4F on peripheral blood cell subpopulations in C57 mice treated with a high fat diet and the mechanism of Rev-D4F in improving the function of EPCs impaired by tumor necrosis factor-α (TNF-α). The high fat diet significantly decreased the number of EPCs, EPC migratory functions, and the percentage of lymphocytes in the white blood cells. However, it significantly increased the number of white blood cells, the percentage of monocytes in the white blood cells, and the level of vascular endothelial growth factor (VEGF) and TNF-α in the plasma. Rev-D4F clearly inhibited the effect of the high fat diet on the quantification of peripheral blood cell subpopulations and cytokine levels, and increased stromal cell derived factor 1α (SDF-1α) in the plasma. We provided in vitro evidence that TNF-α impaired EPC proliferation, migration, and tube formation through inactive AKT and eNOS, which was restored by Rev-D4F treatment. In contrast, both the PI3-kinase (PI3K) inhibitor (LY294002) and AKT inhibitor (perifosine) obviously inhibited the restoration of Rev-4F on EPCs impaired by TNF-α. Our results suggested that Rev-D4F increases the quantity of endothelial progenitor cells through increasing the SDF-1α levels and decreasing the TNF-α level of peripheral blood in high fat diet-induced C57BL/6J mice, and restores TNF-α induced dysfunctions of EPCs partly through stimulating the PI3K/AKT signal pathway.
Topics: Animals; Cardiovascular Diseases; Cells, Cultured; Chemokine CXCL12; Diet, High-Fat; Drug Evaluation, Preclinical; Endothelial Progenitor Cells; Mice, Inbred C57BL; Nitric Oxide Synthase Type III; Peptides; Proto-Oncogene Proteins c-akt; Tumor Necrosis Factor-alpha
PubMed: 26398523
DOI: 10.1371/journal.pone.0138832 -
Food and Chemical Toxicology : An... Jan 2018Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have...
The sp-iminosugar glycolipid 1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO-ONJ) as selective anti-inflammatory agent by modulation of hemeoxygenase-1 in Bv.2 microglial cells and retinal explants.
Neuroinflammation is an early event during diabetic retinopathy (DR) that impacts the dynamics of microglia polarization. Gliosis is a hallmark of DR and we have reported the beneficial effects of 1R-DSO-ONJ, a member of the sp-iminosugar glycolipid (sp-IGL) family, in targeting microglia and reducing gliosis in diabetic db/db mice. Herein, we analyzed the effect of DSO-ONJ, another family compound incorporating a sulfone group that better mimics the phosphate group of phosphatidylinositol ether lipid analogues (PIAs), in Bv.2 microglial cells treated with bacterial lipopolysaccaride (LPS) and in retinal explants from db/db mice. In addition to decreasing iNOS and inflammasome activation, the anti-inflammatory effect of DSO-ONJ was mediated by direct p38α MAPK activation. Computational docking experiments demonstrated that DSO-ONJ binds to p38α MAPK at the same site where PIAs and the alkyl phospholipid perifosine activators do, suggesting similar mechanism of action. Moreover, treatment of microglial cells with DSO-ONJ increased both heme-oxygenase (HO)-1 and Il10 expression regardless the presence of LPS. In retinal explants from db/db mice, DSO-ONJ also induced HO-1 and reduced gliosis. Since IL-10-mediated induction of HO-1 expression is mediated by p38α MAPK activation, our results suggest that this molecular mechanism is involved in the anti-inflammatory effects of DSO-ONJ in microglia.
Topics: Animals; Anti-Inflammatory Agents; Cell Line; Gene Expression Regulation, Enzymologic; Glycolipids; Heme Oxygenase-1; Lipopolysaccharides; Mice; Mice, Inbred NOD; Microglia; Retina; Tissue Culture Techniques
PubMed: 29191728
DOI: 10.1016/j.fct.2017.11.050 -
Beijing Da Xue Xue Bao. Yi Xue Ban =... Feb 2018To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism.
[Decreased phosphorylation of mitogen activated protein kinase and protein kinase B contribute to the inhibition of osteogenic differentiation mediated by activation of Toll like receptor in human periodontal ligament stem cells].
OBJECTIVE
To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism.
METHODS
Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium.
RESULTS
Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression.
CONCLUSION
Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.
Topics: Cell Differentiation; Cells, Cultured; Humans; Ligaments; Mitogen-Activated Protein Kinases; Osteogenesis; Periodontal Ligament; Phosphorylation; Proto-Oncogene Proteins c-akt; Stem Cells; Toll-Like Receptors
PubMed: 29483719
DOI: No ID Found -
Cancer Prevention Research... Oct 2016Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor...
Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor suppressor PTCH1 and aberrant activation of Hedgehog (Hh) signaling. Therapies targeted at components of the Hh pathway, including the smoothened (SMO) inhibitor vismodegib, can ablate these tumors clinically, but tumors recur upon drug discontinuation. Using SKH1-Ptch1 as a model that closely mimics the spontaneous and accelerated growth pattern of BCCs in patients with BCNS, we show that AKT1, a serine/threonine protein kinase, is intrinsically activated in keratinocytes derived from the skin of newborn Ptch1 mice in the absence of carcinogenic stimuli. Introducing Akt1 haplodeficiency in Ptch1 mice (Akt1 Ptch1) significantly abrogated BCC growth. Similarly, pharmacological inhibition of AKT with perifosine, an alkyl phospholipid AKT inhibitor, diminished the growth of spontaneous and UV-induced BCCs. Our data demonstrate an obligatory role for AKT1 in BCC growth, and targeting AKT may help reduce BCC tumor burden in BCNS patients. Cancer Prev Res; 9(10); 794-802. ©2016 AACR.
Topics: Animals; Basal Cell Nevus Syndrome; Carcinoma, Basal Cell; Cell Line; Disease Models, Animal; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Proto-Oncogene Proteins c-akt; Skin Neoplasms
PubMed: 27388747
DOI: 10.1158/1940-6207.CAPR-16-0066