GLI2 promotes cell proliferation and migration through transcriptional activation of ARHGEF16 in human glioma cells

Abstract Background The Hedgehog (Hh) signaling pathway plays critical roles in modulating embryogenesis and maintaining tissue homeostasis, with glioma-associated oncogene (GLI) transcription factors being the main mediators. Aberrant activation of this pathway is associated with various human malignancies including glioblastoma, although the mechanistic details are not well understood. Methods We performed a microarray analysis of genes that are differentially expressed in glioblastoma U87 cells overexpressing GLI2A, the active form of GLI2, relative to the control cells. Chromatin immunoprecipitation and dual-luciferase assays were used to determine whether Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a downstream target of GLI2. Then, transwell migration, EdU and soft-agar colony formation assays were employed to test effects of ARHGEF16 on glioma cancer cell migration and proliferation, and the effects of GLI2/ARHGEF16 signaling on tumor growth were examined in vivo. Finally, we performed yeast two-hybrid assay, Co-IP and GST-pull down to identify factors that mediate effects of ARHGEF16. Results We found that ARHGEF16 mRNA level was upregulated in U87 cells overexpressing GLI2A relative to control cells. GLI2 binds to the ARHGEF16 promoter and activates gene transcription. Glioma cells U87 and U118 overexpressing ARHGEF16 showed enhanced migration and proliferation relative to the control cells, while knockdown of ARHGEF16 in H4 cells led to decreased cell proliferation compared to the control H4 cells. In contrast to the promoting effect of GLI2A overexpression on glioma xenograft growth, both GLI2 inhibition and ARHGEF16 knockdown retarded tumor growth. Cytoskeleton-associated protein 5 (CKAP5) was identified as an interaction protein of ARHGEF16, which is important for the stimulatory effects of ARHGEF16 on glioma cell migration and proliferation. Conclusions These results suggest that therapeutic strategies targeting the GLI2/ARHGEF16/CKAP5 signaling axis could inhibit glioma progression and recurrence

Early Predictors of the Increase in Perihematomal Edema Volume After Intracerebral Hemorrhage: A Retrospective Analysis From the Risa-MIS-ICH Study

Asthma has become a global health issue, suffering more than 300 million people in the world, which is a heterogeneous disease, usually characterized by chronic airway inflammation and airway hyperreactivity. Combination of inhaled corticosteroids (ICS) and long acting β-agonists (LABA) can relieve asthma symptoms and reduce the frequency of exacerbations, especially for patients with refractory asthma, but there are limited treatment options for people who do not gain control on combination ICS/LABA. The increase in ICS dose generally provides little additional benefit, and there is an increased risk of side effects. Therefore, therapeutic interventions integrating the use of different agents that focus on different targets are needed to overcome this set of diseases. Some findings suggest autophagy is closely correlated with the severity of asthma through eosinophilic inflammation, and its modulation may provide novel therapeutic approaches for severe allergic asthma. The chinese herbal medicine (CHM) have been demonstrated clinically as potent therapeutic interventions for asthma. Moreover some reports have found that the bioactive components isolated from CHM could modulate autophagy, and exhibit potent Anti-inflammatory activity. These findings have implied the potential for CHMs in asthma or allergic inflammation therapy via the modulation of autophagy. In this review, we discuss the basic pathomechanisms underpinning asthma, and the potential role of CHMs in treating asthma with modulating autophagy

Transcriptional insights of citrus defense response against Diaporthe citri

Abstract Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6’H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism

ITGB4-FAK signaling mediates Shh-induced cellular migration and invasion.

<p>(<b>A, B</b>) Hh signaling activates FAK through ITGB4. SKOV3 cells were treated with ITGB4 antibody or control IgG in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Expression and phosphorylation of FAK (Tyr397) were verified by Western blot. (<b>C–E</b>) Blockade of Gli down-regulates phosphorylation levels of FAK (Tyr397) but not FAK protein expression. SKOV3 cells were treated with GANT61 or DMSO for 24 hr. Then Western blot was performed using the indicated antibodies. N.S, no significance. (<b>F, G</b>) Repression Gli expression decreases the phosphorylation of FAK (Tyr397) and cytoskeletal organization. SKOV3 cells were treated with GANT61 for 48 hr. Subsequently, cells were stained with antibody against phosphorylation of FAK (Tyr397) and visualized using confocal microscope (<b>F</b>). For F-Actin staining, cells were incubated with Alexa Fluor 488 phalloidin (Invitrogen, A12379) for 20 min and visualized using confocal microscope (<b>G</b>). (<b>H</b>) Loss of Gli-FAK signaling impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with control miRNAi or Gli1 miRNAi (miR-Gli1-720) for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) together with PF573228 (5 µM), a specific inhibitor of FAK, or control vehicle (DMSO) for another 24 hr. Cell invasion was measured by invasion assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups. Scale bar, 20 µm.</p

Gene expression profiles in GANT61-treated SKOV3 cells.

<p>(<b>A</b>) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. Changes in gene expression were determined by cDNA microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. (<b>B</b>) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant <i>P</i>-values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log (<i>P</i>-value). (<b>C</b>) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. (<b>D</b>) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a> using the primer sets in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#pone-0088386-t001" target="_blank"><b>Table 1</b></a>. The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.</p

Primers used for real-time PCR amplification.

<p>Primers used for real-time PCR amplification.</p

Down-regulation of Gli decreased ovarian cancer cell migration and invasion.

<p>(<b>A</b>) Exposure to GANT61 (30 µM; 48 hr) reduces expression of both Gli1 and Gli2 protein, determined by Western blot. GAPDH was used as the loading control. (<b>B</b>) Expression of both Gli1 and Gli2 mRNA is decreased following treatment with GANT61 (30 µM; 48 hr), determined by real-time PCR. (<b>C, D</b>) GANT61 inhibits expression of Gli2 in ovarian cancer cells. ES2 and SKOV3 cells were treated with control vehicle (DMSO) or GANT61 (20 µM) for 48 hr. Subsequently, cells were stained with Gli2 and visualized under a confocal microscope. Scale bar, 10 µm. (<b>E, F</b>) Blockade of Hh signaling inhibits ovarian cancer cell migration. Confluent ES2 cell monolayers were wounded with a pipette tip and then treated with N-Shh conditional medium plus control vehicle (0.2% DMSO) or GANT61. Cell migration to the wound area was monitored by microscopy. The percentage of total area covered by cells was then assessed using the NIH Image program. (<b>G, H</b>) Blockade of Hh signaling inhibits ovarian cancer cell invasion. SKOV3 cells (1×10⁵ cells/well) were seeded in the upper chamber. Growth medium containing N-Shh (0.5 µg/ml) alone or together with GANT61 (20 µM), control vehicle (0.2% DMSO) were added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. The representative images from three independent experiments are shown. Scale bar, 200 µm. The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups.</p

Effect of Gli blockade on growth of human ovarian cancer cells <i>in vivo</i>.

<p>SKOV3 cells (2×10⁷) were implanted s.c. into the flank of BALB/c nu/nu mice. Afterwards the mice were received either GANT61 (25 mg/kg s.c., thrice per week) or solvent (corn oil: ethanol, 4∶1). Treatment was initiated in all groups (n = 8 each group) when mean tumor volume had reached 100 mm³. (<b>A</b>) Treatment with GANT61 led to a significant growth inhibition of xenografted human ovarian cancer tumors. n = 8; **, <i>P</i><0.01. (<b>B</b>) The experiment was terminated on day 15, and tumors were excised. Final tumor weights in the GANT61 treatment group (n = 8) were significantly lower compared with excised tumors of the solvent control group (n = 8). **, <i>P</i><0.01. Columns, mean; bars, SD. (<b>C, D</b>) Western blot analyses for Gli1 and Gli2 in tumor tissues. Treatment with GANT61 diminished expression of Gli1 and Gli2. (<b>E, F</b>) Immunohistochemical image of tumor tissue section. Treatment with GANT61 diminished expression of ITGB4 (<b>E</b>). A substantial decrease in phosphorylation of FAK (Tyr397) was also effectively achieved by GANT61 treatment (<b>F</b>). Scale bar, 20 µm.</p

ITGB4 mediates Shh-induced ovarian cancer cell invasion.

<p>(<b>A, B</b>) Stimulation of N-Shh up-regulates ITGB4 expression. SKOV3 cells were incubated in the presence or absence of N-Shh (0.5 µg/ml) for 24 hr. Western blots were then performed for the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. (<b>C, D</b>) Inhibition of Gli1 down-regulates ITGB4 expression. SKOV3 cells were treated with GANT61 or DMSO for 48 hr. Western blot was then performed with the indicated antibodies. Protein expression was quantified by Image J and normalized to GAPDH. (<b>E, F</b>) Blockade of ITGB4 inhibits cell invasion in SKOV3 cells. SKOV3 cells grown in low serum (2% FBS) growth medium were seeded into the upper chambers. The low serum growth medium containing N-Shh (0.5 µg/ml) together with anti-ITGB4 antibody (0.2 µg/ml) or control IgG was added to the lower chamber. After 24 hr of incubation, cells that invaded the lower surface of the insert were stained with crystal violet and counted by microscopy. (<b>G</b>) Gli1 miRNAi inhibits the expression of ITGB4 in SKOV3 cells. SKOV3 cells were transiently transfected with vehicle (lipofectamine alone), control miRNAi and Gli1 miRNAis (miR-Gli1-720 and miR-Gli1-1863), respectively. Western blot showed that Gli1 miRNAi inhibited the expression of ITGB4 genes. (<b>H</b>) Silencing Gli1 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with Gli1 miRNAi or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Cell invasion was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a>. (<b>I, J</b>) Silencing ITGB4 expression impairs Shh-induced ovarian cancer cell invasion. SKOV3 cells were transfected with ITGB4 miRNAi constructs (miR-ITGB4-2255 and miR-ITGB4-4027) or control miRNAi for 24 hr followed by stimulation with N-Shh (0.5 µg/ml) or control medium for another 24 hr. Western blot showed that ITGB4 miRNAis inhibited the expression of ITGB4 gene (<b>I</b>). Cell invasion was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088386#s4" target="_blank">Methods</a> (<b>J</b>). The data are expressed as mean ± SD for experiments performed three times. **, <i>P<</i>0.01, compared with control groups.</p