Silencing of the circ-Arhgap5 RNA protects neuronal PC12 cells against injury and depends on the miR-29a-3p/Rock1 axis.

Circular RNAs (circRNAs) are abundantly expressed in human central nervous system. Here, we explored the role of circ_Arhgap5 in cerebral ischemia/reperfusion (I/R)-induced nerve injury in PC12 cells and its associated mechanism. Cell proliferation ability was assessed by 5-Ethynyl-2'-deoxyuridine (Edu) assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was applied to assess cell apoptotic rate. Cell death was analyzed by lactate dehydrogenase (LDH) assay. Oxygen-glucose deprivation and reoxygenation (OGD/R) up-regulates the expression of circRNA Rho GTPase activating protein 5 (circ_Arhgap5; mmu_circ_0000377) in PC12 cells. OGD/R exposure inhibited the proliferation and induced the apoptosis of PC12 cells, and the silence of circ_Arhgap5 attenuated OGD/R-induced injury in PC12 cells. miR-29a-3p was identified as a target of circ_Arhgap5 in PC12 cells. Circ_Arhgap5 knockdown-mediated protective effects in OGD/R-induced PC12 cells were reversed by the interference of miR-29a-3p. miR-29a-3p interacted with the 3' untranslated region (3'UTR) of Rho-associated coiled-coil-containing protein kinase 1 (Rock1), and circ_Arhgap5 can positively regulate Rock1 expression by sponging miR-29a-3p in PC12 cells. miR-29a-3p overexpression protected PC12 cells against OGD/R-induced damage by down-regulating Rock1. In conclusion, circ_Arhgap5 silencing protected PC12 cells from OGD/R-induced injury through mediating miR-29a-3p/Rock1 axis.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

DOCK4 regulates ghrelin production in gastric X/A-like cells.

PURPOSE: Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. Ghrelin also modulates neuroinflammatory and apoptotic processes. Dedicator of cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor (GEF), is involved in the regulation of neuronal polarization and axon regeneration. However, the effect of DOCK4 on ghrelin production has not been explored. METHODS: The expression of DOCK4 in human and mouse stomach was examined by immunohistochemical staining. The synthesis and secretion of ghrelin in Dock4 null mice were evaluated by real-time quantitative PCR, Western blot and ELISA. The effects of DOCK4 on ghrelin production in mHypoE-42 cells were measured by real-time quantitative PCR and Western blot. RESULTS: We showed that DOCK4 was expressed in both human and mouse gastric ghrelin cells. The mRNA and protein levels of gastric ghrelin, as well as ghrelin secretion, were remarkably diminished in Dock4 null mice. Furthermore, we showed that overexpression of Dock4 significantly stimulated ghrelin expression, while siRNA knockdown of endogenous Dock4 resulted in a marked decrease of ghrelin in mHypoE-N42 cells. CONCLUSIONS: Our results identify DOCK4 as a critical regulator for ghrelin production in gastric X/A-like cells.

Exosomes derived from human umbilical cord mesenchymal stem cells reduce tendon injuries via the miR-27b-3p/ARHGAP5/RhoA signaling pathway.

Tendon injuries are common clinical issues resulted from tissue overuse and age-related degeneration. Previous sutdies have suggested that exosomes secreted by mesenchymal stem cells (MSCs) contribute to tissue injury repair. Here, we provide evidence for a critical role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in reducing tendon injury by activating the RhoA signaling. Treatment of primary injured tenocytes with hucMSC exosomes increases cell proliferation and invasion, which correlates with increased RhoA activity. RhoA mediates the effects of hucMSC exosomes, as treatment of primary injured tenocytes with the RhoA inhibitor, CCG-1423, abolishes the effects of hucMSC exosomes on cell proliferation and invasion. Mechanistically, we observe that hucMSC exosomes induce the expression of a microRNA, miR-27b-3p, which targets and suppresses ARHGAP5, a negative regulator of RhoA. Consistent with this observation, ARHGAP5 overexpression suppresses the effects of hucMSC exosomes on cell proliferation and invasion, while knockdown of ARHGAP5 rescues these effects. Finally, we demonstrate the functional significance of our findings using an Achilles tendon injury model and show that treatment with exosomes reduces tendon injury in rats, which correlates with increased RhoA activity and reduced ARHGAP5 expression. Taken together, our findings highlight a critical role of hucMSC exosomes in reducing tendon injury via miR-27b-3p-mediated suppression of ARHGAP5, resulting in RhoA activation, and leading to increased cell proliferation and invasion of primary injured tenocytes.

Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38ß and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

A WXW motif is required for the anticancer activity of the TAT-RasGAP317-326 peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells.

This study was undertaken to reveal the mechanisms by which RLIP76 regulates endothelial cell angiogenic responses. RLIP76 is an effector of the angiogenic modulator, R-Ras. RLIP76 is overexpressed in many tumors, required for tumor angiogenesis, and blockade of RLIP76 results in tumor regression in multiple models. We report here that RLIP76 was required for expression and secretion of vascular endothelial growth factor (VEGF) in carcinoma and melanoma cells. Conditioned medium derived from RLIP76-depleted tumor cells, but not control knockdown cells, could not stimulate proliferation, migration, or Matrigel cord formation in endothelial cell cultures, which indicates that RLIP76 regulates angiogenic components of the tumor cell secretome. Recombinant VEGF added to conditioned medium from RLIP76-knockdown tumor cells restored these endothelial cell functions. Transcriptional activity of hypoxia-inducible factor 1 (HIF-1), which drives VEGF expression, was blocked in RLIP76-depleted tumor cells. RLIP76 was required for PI3-kinase activation, known to regulate HIF-1, in these cells. However, HIF-1α expression and nuclear localization were unaffected by RLIP76 knockdown, which suggests that RLIP76 regulates HIF-1 at the functional level. Thus, RLIP76 regulates tumor cell transactivation of endothelial cells via control of VEGF expression and secretion, providing a new important link in the mechanism of tumor cell induction of angiogenesis.

TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway.

Alpha lipoic acid treatment in late middle age improves cognitive function: Proteomic analysis of the protective mechanisms in the hippocampus.

Alpha lipoic acid (ALA), a powerful antioxidant, has the potential to relieve age-related cognitive impairment and neurodegenerative disease. Clinical randomized controlled studies have demonstrated the cognitive improvement effects of lipoic acid in Alzheimer's disease (AD). In the present study, we examined the effects of ALA on cognitive function in ageing mice and its protective mechanisms. Eighteen-month-old male C57BL6/J mice received ALA or normal saline for 2 months. The Morris water maze test revealed improved cognitive function in animals that received ALA. Furthermore, tandem Mass Tags (TMT) based liquid chromotography with mass spectrometry/mass spectrometry (LC-MS/MS) was established to identify the target proteins. The results showed that 10 proteins were changed significantly. Gene Ontology (GO) analysis indicated that the upregulated proteins were enriched in terminal bouton, synaptic transmission and lipid transporter activity while the down-regulated proteins were involved in nuclear transcription factor-κB binding, apoptosis and mitogen-activated protein kinase binding. Based on the GO results, two upregulated proteins oxysterol-binding protein-related protein 10 (OSBPL10) and oligophrenin 1 (OPHN1), and one downregulated protein, CDK5 regulatory subunit-associated protein 3 (CDK5rap3), were validated through Western blotting. The results were consistent with the proteomic results. Modulation of synaptic transmission, lipid transporter activity and neuroinflammation appears to be the mechanisms of ALA in the aged brain.

ARHGAP9 inhibits colorectal cancer cell proliferation, invasion and EMT via targeting PI3K/AKT/mTOR signaling pathway.

AIM: In digestive system, colorectal cancer (CRC) is a common malignant tumor. The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway plays a central role in CRC, and the aberrant activation of this pathway is associated with tumorigenesis. We aimed to explore the role of Rho GTPase activating protein 9 (ARHGAP9) in the progression of CRC as well as its regulatory effects on the PI3K/AKT/mTOR pathway. METHODS: The expression of ARHGAP9 in CRC tumor tissues and cell lines were detected using reverse transcription-quantitative PCR (qRT-PCR). 5-ethynyl-2'-deoxyuridine (EdU) assay was applied to test the cell proliferation. Cell migration and invasion were both assessed through transwell assay. Xenograft mouse models were constructed to explore the effects of ARHGAP9 on CRC in vivo. The expressions of PI3K/AKT/mTOR-activating factors and epithelial-mesenchymal transition (EMT)-related factors were all determined using western blot. LY294002 was employed to block PI3K/AKT/mTOR pathway in CRC cells. RESULTS: The expression of ARHGAP9 was down-regulated in CRC tumor tissues and cell lines when compared to normal tissues and cells. The over-expression of ARHGAP9 inhibited cell proliferation, invasion, migration and EMT in CRC cell lines while the knockdown of ARHGAP9 promoted them. In addition, ARHGAP9 up-regulation inhibited the activation of PI3K/AKT/mTOR signaling pathway in CRC cell lines while ARHGAP9 down-regulation led to an opposite effect. The over-expression of ARHGAP9 suppressed CRC tumor growth in vivo. When the PI3K/AKT/mTOR pathway was blocked in CRC cells, the effects of ARHGAP9 knockdown on cell proliferation, migration, invasion and EMT were all overturned. CONCLUSION: ARHGAP9 inhibited the malignant phenotypes of CRC cells via interdicting PI3K/AKT/mTOR signaling pathway.