Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Functional signaling of membrane-bound TL1A induces IFN-γ expression

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4⁺ T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-γ that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-γ secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.     

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-β (TGF-β). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-β-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-β-induced apoptosis.     

Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-γ and p53 signaling

Interferon-γ (IFN-γ) engenders strong antiproliferative responses, in part through activation of p53. However, the long-known IFN-γ-dependent upregulation of human Trp-tRNA synthetase (TrpRS), a cytoplasmic enzyme that activates tryptophan to form Trp-AMP in the first step of protein synthesis, is unexplained. Here we report a nuclear complex of TrpRS with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and with poly(ADP-ribose) polymerase 1 (PARP-1), the major PARP in human cells. The IFN-γ-dependent poly(ADP-ribosyl)ation of DNA-PKcs (which activates its kinase function) and concomitant activation of the tumor suppressor p53 were specifically prevented by Trp-SA, an analog of Trp-AMP that disrupted the TrpRS-DNA-PKcs-PARP-1 complex. The connection of TrpRS to p53 signaling in vivo was confirmed in a vertebrate system. These and further results suggest an unexpected evolutionary expansion of the protein synthesis apparatus to a nuclear role that links major signaling pathways.     

Apigenin suppresses TGF-β1-induced cardiac fibroblast differentiation and collagen synthesis through the downregulation of HIF-1α expression by miR-122-5p

BackgroundApigenin can reduce cardiomyocyte hypertrophy by downregulating hypoxia inducible factor-1 alpha (HIF-1α) expression. However, its effects on cardiac fibroblasts (CFs) and its exact inhibitory molecular mechanisms on HIF-1α remain unclear.PurposeThis study aims to examine the effects of apigenin on cell proliferation and differentiation, microRNA-122-5p (miR-122-5p) expression, and HIF-1α-mediated Smad signaling pathway in transforming growth factor beta 1 (TGF-β1)-stimulated CFs and cardiac fibrosis and to investigate the relationship between miR-122-5p and HIF-1α.MethodsThe TGF-β1-stimulated CFs, the combination of TGF-β1-stimulated and miR-122-5p mimic-transfected CFs, the combination of TGF-β1-stimulated and miR-122-5p inhibitor-transfected CFs, and the isoproterenol-induced cardiac fibrotic mice were used and treated with or without apigenin. The recombinant lentiviruses overexpressing HIF-1α vector and miR-122-5p mimic were co-transfected to observe their interaction. Related mRNA and protein expressions and myocardial collagen were determined. The luciferase reporter gene that contains HIF-1α wild type or mutant type 3’-UTR was used, and the luciferase activity was determined to verify the direct link between miR-122-5p and HIF-1α.ResultsIn the TGF-β1-stimulated CFs, apigenin treatment increased the miR-122-5p and Smad7 expressions and decreased the HIF-1α, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 expressions. Similar and inverse results were observed in the miR-122-5p mimic- and inhibitor-transfected CFs, respectively. Moreover, the miR-122-5p mimic could antagonize the effects of TGF-β1 in the TGF-β1 and miR-122-5p mimic-combined CFs, and the miR-122-5p inhibitor could enhance the effects of TGF-β1 in the TGF-β1 and miR-122-5p inhibitor-combined CFs. In the two aforementioned cell models, the addition of apigenin could further enhance the effects of miR-122-5p mimic and partially reverse the effects of miR-122-5p inhibitor. After treatment of HIF-1α-transfected CFs with miR-122-5p mimic, the HIF-1α expression decreased. Further study confirmed that HIF-1α was a direct target of miR-122-5p. Apigenin also decreased the myocardial collagen accumulation in cardiac fibrotic mice.ConclusionApigenin could suppress the differentiation and collagen synthesis of TGF-β1-stimulated CFs and mouse cardiac fibrosis, and its mechanisms were related to the increment of miR-122-5p expression and subsequent downregulation of HIF-1α expression via direct interaction, which might finally result in the decrements of Smad2/3 and p-Smad2/3 expressions and increment of Smad7 expression.     

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.     

Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch

Transforming growth factor-β (TGF-β) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-β induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-β caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-β-induced apoptosis. However, vNox4, but not TGF-β, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-β enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-β-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.