CCN2-induced lymphangiogenesis is mediated by the integrin αvβ5-ERK pathway and regulated by DUSP6.
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ABSTRACT:
SUBMITTER: Hashiguchi S
PROVIDER: S-EPMC8766563 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHigh tidal volume ventilation of healthy lungs or exacerbation of existing acute lung injury (ALI) by more moderate mechanical ventilation (MTV) produces ventilator-induced lung injury. It is less clear whether extrapulmonary sepsis sensitizes the lung to MTV.MethodsWe used a two-hit model of cecal ligation and puncture (CLP) followed 12 h later by MTV (10 ml/kg; 6 h) to determine whether otherwise noninjurious MTV enhances CLP-induced ALI by contrasting wildtype and TLR4-/- mice with respect to: alveolar-capillary permeability, histopathology and intrapulmonary levels of WNT-inducible secreted protein 1 (WISP1) and integrin β5; plasma levels of cytokines and chemokines (TNF-α, IL-6, MIP-2, MCP-1) and intrapulmonary neutrophil infiltration; and other inflammatory signaling via intrapulmonary activation of JNK, p38 and ERK. A separate cohort of mice was pretreated with intratracheal neutralizing antibodies to WISP1, integrin β5 or IgG as control and the presented phenotyping repeated in a two-hit model; there were 10 mice per group in these first three experiments. Also, isolated peritoneal macrophages (PM) from wildtype and TLR4-/-, MyD88-/- and TRIF-/- mice were used to identify a WISP1-TLR4-integrin β5 pathway; and the requisite role of integrin β5 in WISP1-induced cytokine and chemokine production in LPS-primed PM was examined by siRNA treatment.ResultsMTV, that in itself did not cause ALI, exacerbated increases in alveolar-capillary permeability, histopathologic scoring and indices of pulmonary inflammation in mice that previously underwent CLP; the effects of this two-hit model were abrogated in TLR4-/- mice. Attendant with these findings was a significant increase in intrapulmonary WISP1 and integrin β5 in the two-hit model. Anti-WISP1 or anti-integrin β5 antibodies partially inhibited the two-hit phenotype. In PM, activation of TLR4 led to an increase in integrin β5 expression that was MyD88 and NF-κB dependent. Recombinant WISP1 increased LPS-induced cytokine release in PM that was inhibited by silencing either TLR4 or integrin β5.ConclusionsThese data show for the first time that otherwise noninjurious mechanical ventilation can exacerbate ALI due to extrapulmonary sepsis underscoring a potential interactive contribution of common events (sepsis and mechanical ventilation) in critical care, and that a WISP1-TLR4-integrin β5 pathway contributes to this phenomenon.
| S-EPMC6240278 | biostudies-literature
Project description:Our previous study has validated that higher matrix stiffness obviously improves vascular endothelial growth factor (VEGF) expression in HCC cells, highlighting a linkage between matrix stiffness and HCC angiogenesis. However, the effects of matrix stiffness on vascular endothelial cells in HCC and its underlying mechanism remain largely uncharacterized. Here we further analyzed the expression of vascular endothelial growth factor receptor 2 (VEGFR2) in human umbilical vein endothelial cells (HUVECs) grown on different stiffness substrates and explored its regulatory mechanism for better understanding matrix stiffness-regulated angiogenesis in HCC. Our results revealed that increased matrix stiffness significantly upregulated the expression of VEGFR2 in HUVECs, and the expression level of VEGFR2 was positively correlated with the expression levels of COL1 and lysyl oxidase in human HCC tissues and rat HCC tissue, moreover VEGFR2 and CD34 were co-localized at blood vessel of HCC tissues, indicating an obvious regulation role of matrix stiffness in VEGFR2 expression. Simultaneously, increased matrix stiffness also elevated the phosphorylation level of Akt and the expressions of integrin αV/β5 and nuclear Sp1 in HUVECs. Inhibition of integrin αVβ5 remarkably reversed the expression of VEGFR2 and phosphorylation level of Akt in HUVECs grown on higher stiffness substrate. Except that, PI3K inhibitor also suppressed the phosphorylation level of Akt and the expressions of VEGFR2 and nuclear Sp1 evidently. Taken together, higher matrix stiffness increased VEGFR2 expression in HUVECs, and integrin αVβ5/Akt/Sp1 pathway participated in stiffness-mediated effects on VEGFR2 upregulation. This study combining with our previous report discloses a new paradigm in which higher matrix stiffness as an initiator drives HCC angiogenesis via upregulating both VEGFR2 expression in vascular endothelial cells and VEGF expression in HCC cells.
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Project description:PurposeTo elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by photoreceptor outer segments (POS) and its effects on retinal pigment epithelium (RPE).MethodsPGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK), and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK) were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS), mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal) after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.ResultsPOS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.ConclusionsThe binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.
| S-EPMC4526642 | biostudies-literature
Project description:Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter.
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Project description:Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvβ5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvβ5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvβ5 is induced during the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF-like molecule 8. In the absence of αvβ5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and IL 10. Mice lacking αvβ5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvβ5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.
| S-EPMC6207937 | biostudies-literature
Project description:Bone morphogenetic protein (BMP)-1 is expressed by odontoblasts in the dentin-pulp complex. Although the functional effects of BMP-1 on the maturation of various preforms of proteins and enzymes involved in initiating mineralization have been widely observed, how BMP-1 affects cellular molecules remains unknown. We performed a comprehensive analysis of BMP-1-altered glycome profiles and subsequent assays to identify the target glycoproteins in human dental pulp cells (hDPCs) by a glycomic approach. In the presence of BMP-1, a lectin microarray analysis and lectin-probed blotting showed that α2,6-sialylation was significantly attenuated in insoluble fractions from hDPCs. Six proteins were identified by a mass spectrometry analysis of α2,6-sialylated glycoproteins purified using a lectin column. Among them, glucosylceramidase (GBA1) was found to accumulate in the nuclei of hDPCs in the presence of BMP-1. Moreover, BMP-1-induced cellular communication network factor (CCN) 2 expression, which is well known as the osteogenesis/chondrogenesis marker, was significantly suppressed in the cells transfected with GBA1 siRNA. Furthermore, importazole, a potent inhibitor of importin-β-mediated nuclear import significantly suppressed BMP-1-induced GBA1 nuclear accumulation and BMP-1-induced CCN2 mRNA expression, respectively. Thus, BMP-1 facilitates the accumulation of GBA1 in the nucleus through the reduction of α2,6-sialic acid, which potentially contributes to the transcriptional regulation of the CCN2 gene via importin-β-mediated nuclear import pathway in hDPCs. Our results offer new insights into the role of the BMP-1-GBA1-CCN2 axis in the development, tissue remodeling, and pathology of dental/craniofacial diseases.
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Project description:Objective: DUSP6 is a negative regulator of the ERK signaling pathway and plays an important role in chemotherapy-resistance. Previously we showed that DUSP6 is overexpressed in ovarian cancer side population (SP) cells that possess cancer stem cell-like properties and are quiescent and chemotherapy-resistant. Here, we explore the effects of DUSP6 on chemotherapy-resistance by examining its regulation of the ERK signaling pathway and G0/G1 cell cycle arrest. Methods: mRNA and protein expression of DUSP6 and G0/G1 cell cycle checkpoint regulating proteins (CyclinD1, CyclinD3 and CyclinE2) was evaluated among ovarian cancer cell lines and tissue samples. Ovarian cancer cells were transiently transfected to overexpress DUSP6. After treatment with cisplatin, cell viability was measured by the MTS assay at 48 hours and the half maximal inhibitory concentration (IC50) for each cell line was calculated. Subcellular localization and cell cycle analysis were determined by using immunofluorescence and FACS, respectively. Results: SKOV3 and OVCAR8 SP cells were shown to express higher levels of DUSP6 and lower levels of CyclinD3 compared with non-SP (NSP) cells (P<0.001). Among 39 ovarian cancer tissue samples, expression of DUSP6 in the chemotherapy-resistant group (12 samples) was higher than in the chemotherapy-sensitive group (27 samples) (P<0.05). While a lower level of expression of CyclinD3 was seen in the chemotherapy-resistant group, it was not statistically different from the chemotherapy-sensitive group. HO8910 cells where shown to have higher IC50 to cisplatin than SKOV3 or OVCAR8 cells, and this correlated with higher levels of DUSP6 expression. Overexpression of DUSP6 in SKOV3 cells led to an increase in cisplatin IC50 values (P<0.05), and also markedly reduced the expression levels of phospho-ERK1/2 and CyclinD3 and to the predominance of cells in the G0/G1 phase. Conclusion: Our findings reveal an enhancement of chemotherapy-resistance and a predominance of cells in G1 cell cycle arrest in DUSP6-overexpressing ovarian cancer cells. This suggests that overexpression of DUSP6 promotes chemotherapy-resistance through the negative regulation of the ERK signaling pathway, increasing the G0/G1 phase ratio among ovarian cancer cells, and leading to cellular quiescence.
| S-EPMC7097933 | biostudies-literature
Project description:Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.
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Project description:Trophoblast dysfunction is one mechanism implicated in the etiology of recurrent miscarriage (RM). Regulation of trophoblast function, however, is complex and the mechanisms contributing to dysregulation remain to be elucidated. Herein, we found EIF5A1 expression levels to be significantly decreased in cytotrophoblasts in RM villous tissues compared with healthy controls. Using the HTR-8/SVneo cell line as a model system, we found that overexpression of EIF5A1 promotes trophoblast proliferation, migration and invasion in vitro. Knockdown of EIF5A1 or inhibiting its hypusination with N1-guanyl-1,7-diaminoheptane (GC7) suppresses these activities. Similarly, mutating EIF5A1 to EIF5A1K50A to prevent hypusination abolishes its effects on proliferation, migration and invasion. Furthermore, upregulation of EIF5A1 increases the outgrowth of trophoblasts in a villous explant culture model, whereas knockdown has the opposite effect. Suppression of EIF5A1 hypusination also inhibits the outgrowth of trophoblasts in explants. Mechanistically, ARAF mediates the regulation of trophoblast migration and invasion by EIF5A1. Hypusinated EIF5A1 regulates the integrin/ERK signaling pathway via controlling the translation of ARAF. ARAF level is also downregulated in trophoblasts of RM villous tissues and expression of ARAF is positively correlated with EIF5A1. Together, our results suggest that EIF5A1 may be a regulator of trophoblast function at the maternal-fetal interface and low levels of EIF5A1 and ARAF may be associated with RM.
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Project description:Minichromosome maintenance 6 (MCM6) has been implicated in the progression of various malignant tumors; however, its exact physiological function in kidney diseases remains unclear. Here, we demonstrated that MCM6 levels showed a significant increase in the proximal tubular cells during progressive renal fibrosis in two unrelated in vivo fibrotic models, including unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI). Depletion of MCM6 aggravated partial epithelial-mesenchymal transition, extracellular matrix accumulation, and myofibroblast activation in the kidneys of UUO or UIRI mice. Conversely, overexpression of MCM6 promoted the recovery of E-cadherin and retarded UUO- or UIRI-induced renal fibrosis. In addition, DUSP6 expression substantially decreased in fibrotic kidneys, and it might be involved in MCM6-induced renal fibrosis by regulating the activation of ERK/GSK-3β/Snail1 signaling. In conclusion, our results highlight the significance of MCM6 in renal fibrosis, providing a potential therapeutic target for patients with chronic kidney disease.
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