scholarly journals Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuangshuang Wang ◽  
Hua Qian ◽  
Liwei Zhang ◽  
Panpan Liu ◽  
Dexuan Zhuang ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Activated T Cells ◽  
Ras Pathway ◽  
Ras Family ◽  
Interfering Rna

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

scholarly journals Phaseolin Attenuates Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells and Zebrafish

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 420
Author(s):  
Su-Jung Hwang ◽  
Ye-Seul Song ◽  
Hyo-Jong Lee
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Raw 264.7 Cells ◽  
Network Pharmacology ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Bioactive Constituents

Kushen (Radix Sophorae flavescentis) is used to treat ulcerative colitis, tumors, and pruritus. Recently, phaseolin, formononetin, matrine, luteolin, and quercetin, through a network pharmacology approach, were tentatively identified as five bioactive constituents responsible for the anti-inflammatory effects of S. flavescentis. However, the role of phaseolin (one of the primary components of S. flavescentis) in the direct regulation of inflammation and inflammatory processes is not well known. In this study, the beneficial role of phaseolin against inflammation was explored in lipopolysaccharide (LPS)-induced inflammation models of RAW 264.7 macrophages and zebrafish larvae. Phaseolin inhibited LPS-mediated production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), without affecting cell viability. In addition, phaseolin suppressed pro-inflammatory mediators such as cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in a dose-dependent manner. Furthermore, phaseolin reduced matrix metalloproteinase (MMP) activity as well as macrophage adhesion in vitro and the recruitment of leukocytes in vivo by downregulating Ninjurin 1 (Ninj1), an adhesion molecule. Finally, phaseolin inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB). In view of the above, our results suggest that phaseolin could be a potential therapeutic candidate for the management of inflammation.

Coordinate expression of secretory phospholipase A2 and cyclooxygenase-2 in activated human keratinocytes

2000 ◽  
Vol 278 (4) ◽  
pp. C822-C833 ◽  
Author(s):  
Krystyna E. Rys-Sikora ◽  
Raymond L. Konger ◽  
John W. Schoggins ◽  
Rama Malaviya ◽  
Alice P. Pentland
Keyword(s):  
Wound Repair ◽  
Human Keratinocytes ◽  
Plating Density ◽  
Coordinate Expression ◽  
Cox 2 ◽  
Type V ◽  
In Vitro Wounding

PGE2 levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE2 production in activated keratinocytes are unclear. In previous studies, we showed that PGE2 is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE2 synthesis is an important component of wound healing. Here, we examine the role of phospholipase A2(PLA2) and cyclooxygenase (COX) enzymes in modulation of PGE2 production. We report that the increased PGE2 production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA2 (sPLA2) proteins. Increased sPLA2 activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA2, along with the M-type sPLA2 receptor. Thus the coordinate expression of sPLA2 and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair.

scholarly journals Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions

2020 ◽  
Vol 105 (5) ◽  
pp. 1316-1326
Author(s):  
Yong Song ◽  
Ren-Wei Su ◽  
Niraj R Joshi ◽  
Tae Hoon Kim ◽  
Bruce A Lessey ◽  
...  
Keyword(s):  
Mouse Model ◽  
Vehicle Group ◽  
E Proteins ◽  
Eutopic Endometrium ◽  
Baboon Model ◽  
Notch1 Signaling Pathway ◽  
Interfering Rna

Abstract Context NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer. Objective To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis. Setting and Design The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo. Results E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model. Conclusions IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development.

scholarly journals MPC1 Deficiency Promotes CRC Liver Metastasis via Facilitating Nuclear Translocation of β-Catenin

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Guang-Ang Tian ◽  
Chun-Jie Xu ◽  
Kai-Xia Zhou ◽  
Zhi-Gang Zhang ◽  
Jian-Ren Gu ◽  
...  
Keyword(s):  
Tumor Metastasis ◽  
Nuclear Translocation ◽  
Tca Cycle ◽  
Mitochondrial Pyruvate Carrier ◽  
Human Sample ◽  
Metastatic Capacity ◽  
Tcga Dataset

Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/β-catenin pathway by promoting nuclear translocation of β-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.

Constitutive TGF-β1 Deficiency Induces a Defect in Hematopoietic Stem/Progenitor Cell Compartment in Fetus and Neonate.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1397-1397
Author(s):  
Claude Capron ◽  
Catherine Lacout ◽  
Yann Lecluse ◽  
Valérie Jalbert ◽  
Elisabeth Cramer Bordé ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Type I ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Tgf Β1

Abstract TGF-β1 is a cytokine with pleiotropic effects. It has been considered that TGF-β1plays a major role on hematopoietic stem cells (HSC) based on in vitro experiment. Achieving in vivo experiments proved to be difficult because constitutive TGF-β1 knock-out (KO) in mice leads to lethality during the first 4 weeks of life from a wasting syndrome related to tissue infiltration by activated T cells and macrophages. For this reason, hematopoiesis of TGF-β1−/− mice has not been studied in details. In contrast the role of TGF-β1 has been recently extensively studied in conditional TGF-β type I receptor (TβRI) KO mice. No clear effect was observed on HSC functions, suggesting that TGF-β1 was not a key physiological regulator of hematopoiesis in the adult. However, these experiments have some limitations. They do not exclude a putative role for TGF-β1 during fetal hematopoiesis and they do not specifically address the role of TGF-β1 on hematopoiesis because KO of TGF-β receptor leads to signaling arrest for all TGF-βs. In addition, other receptors may be involved in TGF-β1 signaling. For these reasons, we have investigated the hematopoiesis of constitutive TGF-β1 KO mice with a mixed Sv129 × CF-1 genetic background allowing the birth of a high proportion of homozygotes. In 2 week-old neonate mice, we have shown a decrease of bone marrow (BM) and spleen progenitors and a decrease of immature progenitors colony forming unit of the spleen (CFU-s). Moreover this was associated with a loss in reconstitutive activity of TGF-β1−/− HSC from BM. However, although asymptomatic, these mice had an excess of activated lymphocytes and an augmentation of Sca-1 antigen on hematopoietic cells suggesting an excess of γ-interferon release. Thus we studied hematopoiesis of 7 to 10 days-old neonate mice, before phenotypic modification and inflammatory cytokine release. Similar results were observed with a decrease in the number of progenitors and in the proliferation of TGF-β1−/− BM cells along with an increased differentiation but without an augmentation in apoptosis. Moreoever, a loss of long term reconstitutive capacity of BM Lineage negative (Lin−) TGF-β1−/− cells along with a diminution of homing of TGF-β1−/− progenitors was found. These results demonstrate that TGF-β1 may play a major role on the HSC/Progenitor compartment in vivo and that this defect does not seem to be linked to the immune disease. To completely overpass the risk of the inflammatory syndrome, we analyzed hematopoiesis of fetal liver (FL) of TGF-β1−/− mice and still found a decrease in progenitors, a profound defect in the proliferative capacities, in long term reconstitutive activity and homing potential of primitive FL hematopoietic cells. Our results demonstrate that TGF-β1 plays an important role during hematopoietic embryonic development. Altogether these findings suggest that TGF-β1 is a potent positive regulator for the in vivo homeostasis of the HSC compartment.

scholarly journals Podocyte-specific knockin of PTEN protects kidney from hyperglycemia

2018 ◽  
Vol 314 (6) ◽  
pp. F1096-F1107 ◽  
Author(s):  
Huizhen Wang ◽  
Ziwei Feng ◽  
Jianteng Xie ◽  
Feng Wen ◽  
Menglei Jv ◽  
...  
Keyword(s):  
Diabetic Kidney Disease ◽  
Therapeutic Strategy ◽  
Renal Protection ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog ◽  
Matrix Expansion ◽  
Interfering Rna

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has proven to be downregulated in podocytes challenged with high glucose (HG), and knockout of PTEN in podocytes aggravated the progression of diabetic kidney disease (DKD). However, whether podocyte-specific knockin of PTEN protects the kidney against hyperglycemia in vivo remains unknown. The inducible podocyte-specific PTEN knockin (PPKI) mice were generated by crossing newly created transgenic loxP-stop- loxP-PTEN mice with podocin-iCreERT2 mice. Diabetes mellitus was induced in mice by intraperitoneal injection of streptozotocin at a dose of 150 mg/kg. In vitro, small interfering RNA and adenovirus interference were used to observe the role of PTEN in HG-treated podocytes. Our data demonstrated that PTEN was markedly reduced in the podocytes of patients with DKD and focal segmental glomerulosclerosis, as well as in those of db/db mice. Interestingly, podocyte-specific knockin of PTEN significantly alleviated albuminuria, mesangial matrix expansion, effacement of podocyte foot processes, and incrassation of glomerular basement membrane in diabetic PPKI mice compared with wild-type diabetic mice, whereas no alteration was observed in the level of blood glucose. The potential renal protection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and motility and the inhibition of apoptosis. Our results showed that podocyte-specific knockin of PTEN protected the kidney against hyperglycemia in vivo , suggesting that targeting PTEN might be a novel and promising therapeutic strategy against DKD.

scholarly journals 4-Acetyl-Antroquinonol B Suppresses SOD2-Enhanced Cancer Stem Cell-Like Phenotypes and Chemoresistance of Colorectal Cancer Cells by inducing hsa-miR-324 re-Expression

Cancers ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 269 ◽  
Author(s):  
Oluwaseun Adebayo Bamodu ◽  
Ching-Kuo Yang ◽  
Wei-Hong Cheng ◽  
David T.W. Tzeng ◽  
Kuang-Tai Kuo ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Aberrant Expression ◽  
Colon Cells ◽  
Colorectal Cancer Cells ◽  
Enhanced Expression ◽  
Polymerase Chain ◽  
Interfering Rna

Background: Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality in both sexes globally. This is not unconnected with the heterogeneity and plasticity of CRC stem cells (CRC-SCs) which stealthily exploit the niche-related and (epi)genetic factors to facilitate metastasis, chemoresistance, tumor recurrence, and disease progression. Despite the accumulating evidence of the role of dysregulated microRNAs in malignancies, the therapeutic efficacy of pharmacological-targeting of CRC-SC-associated microRNAs is relatively under-explored. Experimental approach: In this present study, we employed relatively new bioinformatics approaches, analyses of microarray data, Western blot, real-time polymerase chain reaction (RT-PCR), and functional assays to show that hsa-miR-324-5p expression is significantly suppressed in CRC cells, and inversely correlates with the aberrant expression of SOD2. Results: This converse hsa-miR-324-5p/SOD2 relationship is associated with enhanced oncogenicity, which is effectively inhibited by 4-acetylantroquinonol B (4-AAQB), as evidenced by inhibited cell viability and proliferation, as well as attenuated migration, invasion, and clonogenicity in 4-AAQB-treated DLD1 and HCT116 cells. Interestingly, 4-AAQB did not affect the viability and proliferation of normal colon cells. We also showed that 4-AAQB-induced re-expression of hsa-miR-324-5p, akin to short-interfering RNA, reduced SOD2 expression, correlates with the concurrent down-regulation of SOD2, N-cadherin, vimentin, c-Myc, and BcL-xL2, with concomitant up-regulation of E-cadherin and BAX2 proteins. Enhanced expression of hsa-miR-324-5p in the CRC cells suppressed their tumorigenicity in vitro and in vivo. Additionally, 4-AAQB synergistically potentiates the FOLFOX (folinate (leucovorin), fluorouracil (5FU), and oxaliplatin) anticancer effect by eliciting the re-expression of SOD2-suppressed hsa-miR-324, and inhibiting SOD2-mediated tumorigenicity. Conclusion: Our findings highlight the pre-clinical anti-CSC efficacy of 4-AAQB, with or without FOLFOX in CRC, and suggest a potential novel therapeutic strategy for CRC patients.

scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

2017 ◽  
Author(s):  
Ajay Mishra ◽  
Angela Oliveira Pisco ◽  
Benedicte Oules ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Stable States ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation1,2. While progress has been made in characterising the stem and differentiated cell compartments3, the transition between the two cell states, termed commitment4, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h. We have previously shown that commitment begins at approximately 4h and differentiation is initiated by 8h5. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal differentiation by negatively regulating ERK MAPK and positively regulating key API transcription factors. Their activity is antagonised by concomitant upregulation of the anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of transient positive and negative interactions, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem cell and differentiated cell) via an unstable (committed) state. In addition phosphatase expression is spatially regulated relative to the location of stem cells, both in vivo and in response to topographical cues in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

scholarly journals Trehalose Alleviates Crystalline Silica-Induced Pulmonary Fibrosis via Activation of the TFEB-Mediated Autophagy-Lysosomal System in Alveolar Macrophages

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 122 ◽  
Author(s):  
Xiu He ◽  
Shi Chen ◽  
Chao Li ◽  
Jiaqi Ban ◽  
Yungeng Wei ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Alveolar Macrophages ◽  
Nuclear Translocation ◽  
Protective Role ◽  
Crystalline Silica ◽  
Autophagic Flux ◽  
Lysosomal System

Silicosis is an occupational lung disease characterized by persistent inflammation and irreversible fibrosis. Crystalline silica (CS) particles are mainly phagocytized by alveolar macrophages (AMs), which trigger apoptosis, inflammation, and pulmonary fibrosis. Previously, we found that autophagy-lysosomal system dysfunction in AMs was involved in CS-induced inflammation and fibrosis. Induction of autophagy and lysosomal biogenesis by transcription factor EB (TFEB) nuclear translocation can rescue fibrotic diseases. However, the role of TFEB in silicosis is unknown. In this study, we found that CS induced TFEB nuclear localization and increased TFEB expression in macrophages both in vivo and in vitro. However, TFEB overexpression or treatment with the TFEB activator trehalose (Tre) alleviated lysosomal dysfunction and enhanced autophagic flux. It also reduced apoptosis, inflammatory cytokine levels, and fibrosis. Both pharmacologically inhibition of autophagy and TFEB knockdown in macrophages significantly abolished the antiapoptotic and anti-inflammatory effects elicited by either TFEB overexpression or Tre treatment. In conclusion, these results uncover a protective role of TFEB-mediated autophagy in silicosis. Our study suggests that restoration of autophagy-lysosomal function by Tre-induced TFEB activation may be a novel strategy for the treatment of silicosis.

scholarly journals RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis

2011 ◽  
Vol 22 (17) ◽  
pp. 3263-3275 ◽  
Author(s):  
T. T. Giang Ho ◽  
Audrey Stultiens ◽  
Johanne Dubail ◽  
Charles M. Lapière ◽  
Betty V. Nusgens ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cancer Progression ◽  
Dynamic Balance ◽  
Cellular Functions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Interfering Rna

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug–activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

Sign in / Sign up

Export Citation Format

Share Document