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BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 μM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Epidermal growth factor receptor (EGFR) plays an important role in gastric cancer (GC) progression. Our previous data demonstrated that type II cGMP-dependent protein kinase (PKG II) could block the EGF-EGFR axis as well as down-stream signaling pathways, for example, MAPK, PI3 K, and PLC in GC cells. However, the exact mechanisms of PKG II against cancer remain unclear. Therefore, the present work was to address the above question. Human GC cell line AGS was infected with adenoviral construct encoding cDNA of PKG II (Ad-PKG II) to up-regulate PKG II and then treated with 8-pCPT-cGMP. Two-dimensional electrophoresis (2-DE) was used to analyze the changes of protein expression in the cells. The results showed that 17 proteins had more than twofold changes in EGF-treated group compared with control. However, Ad-PKG II could effectively reversed the changes. Furthermore, far upstream element-binding protein 1 (FUBP1) and MarvelD3 were chosen and PKG II activation reversed EGF/EGFR-induced up-regulation of FUBP1 and downregulation of MarvelD3, respectively. MarvelD3 silence effectively abolished the inhibitory effect of PKG II on EGF-triggered migration. These data indicated that the inhibitory effect of PKG II partially was associated with MarvelD3., (© 2017 International Federation for Cell Biology.)

Non-small cell lung cancer (NSCLC) remains one of the most metastasizing tumors, and directional cell migration is critical for targeting tumor metastasis. GIT2 has been known to bind to Paxillin to control cell polarization and directional migration. However, the molecular mechanisms underlying roles of GIT2 in controlling cell polarization and directional migration remain elusive. Here we demonstrated GIT2 control cell polarization and direction dependent on the regulation of Golgi through RUSC2. RUSC2 interacts with SHD of GIT2 in various lung cancer cells, and stabilizes GIT2 (Mazaki et al., 2006; Yu et al., 2009) by decreasing degradation and increasing its phosphorylation. Silencing of RUSC2 showed reduced stability of GIT2, defective Golgi reorientation toward the wound edge and decreased directional migration. Moreover, short-term EGF stimulation can increase the interaction between RUSC2 and GIT2, prolonged stimulation leads to a decrease of their interaction through activating Rab35. Silencing of Rab35 also reduced stability and phosphorylation of GIT2 and decreased cell migration. Taken together, our study indicated that RUSC2 participates in EGFR signaling and regulates lung cancer progression, and may be a new therapeutic target against lung cancer metastasis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Previous research has demonstrated that type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF)‑initiated signal transduction of MAPK‑mediated, PI3K/Akt‑mediated and PLCγ1‑mediated pathways through blocking EGF‑induced phosphorylation/activation of EGF receptor (EGFR). As EGF/EGFR signaling also initiated signal transduction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT)‑mediated pathway, the present study was performed to investigate whether PKG II exerts an inhibitory effect this pathway. AGS human gastric cancer cell line was infected with adenoviral constructs encoding the cDNA of PKG II (Ad‑PKG II), to increase the expression of PKG II, and treated with 8‑pCPT‑cGMP to activate the kinase. Western blotting was performed to detect the phosphorylation/activation of EGFR, JAK1, JAK2, STAT1 and STAT3 and the expression of cell cycle‑associated proteins, including cyclin D1 and cyclin E. EGF‑induced cell cycle changes were detected by flow cytometry. Transcriptional activity was determined by a reporter gene assay. The results demonstrated that EGF treatment increased the phosphorylation of EGFR, JAK1, JAK2, STAT1 and STAT3, increased the expression levels of cyclin D1 and cyclin E, promoted the cells to enter S phase, and stimulated transcriptional activity in the cells. Increased PKG II activity through infecting the cells with Ad‑PKG II and activating the kinase with 8‑pCPT‑cGMP efficiently reversed the changes caused by EGF. The results suggest that PKG II inhibits EGF‑induced signal transduction of the JAK/STAT‑mediated pathway and further confirms that PKG II may be a cancer inhibitor.

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

Background: Our previous research results showed that Type II cGMP dependent protein kinase (PKG II) could block the activation of epidermal growth factor receptor (EGFR) and consequently inhibit the proliferation and the related MAPK/ERK-mediated signal transduction of gastric cancer cell line BGC-823, suggesting that PKG II might inhibit other EGFR-triggered signal transduction pathways and related biological activities of gastric cancer cells. This paper was designed to investigate the potential inhibition of PKG II on EGF/EGFR-induced migration activity and the related signal transduction pathways., Methodology/principal Findings: In gastric cancer cell line AGS, expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA (Ad-PKG II) and treating the cells with cGMP analogue 8-pCPT-cGMP. Phosphorylation of proteins was detected by Western Blotting and active small G protein Ras and Rac1 was measured by "Pull-down" method. Cell migration activity was detected with trans-well equipment. Binding between PKG II and EGFR was detected with Co-IP. The results showed EGF stimulated migration of AGS cell and the effect was related to PLCγ1 and ERK-mediated signal transduction pathways. PKG II inhibited EGF-induced migration activity and blocked EGF-initiated signal transduction of PLCγ1 and MAPK/ERK-mediated pathways through preventing EGF-induced Tyr 992 and Tyr 1068 phosphorylation of EGFR. PKG II bound with EGFR and caused threonine phosphorylation of it., Conclusion/significance: Our results systemically confirms the inhibition of PKG II on EGF-induced migration and related signal transduction of PLCγ1 and MAPK/ERK-mediated pathways, indicating that PKG II has a fargoing inhibition on EGF/EGFR related signal transduction and biological activities of gastric cancer cells through phosphorylating EGFR and blocking the activation of it.

Our previous study found that Type II cGMP-dependent protein kinase (PKG II) is expressed at lower levels in human gastric cancer tissues and cell lines and increasing the expression and activity of PKG II inhibited the proliferation of cancer cell line BGC-823. However, the mechanism through which PKG II inhibits proliferation of gastric cancer cells is still not clear. Herein, we show that PKG II can inhibit EGF-induced MAPK signal transduction. In the gastric cancer cell line BGC-823, the expression and activity of PKG II were increased by infecting the cells with adenoviral construct encoding PKG II cDNA and treating the cells with the cGMP analogue 8-pCPT-cGMP. We found that PKG II inhibited the EGF-induced dual phosphorylation of ERK, a key component of the MAPK signal transduction pathway. Upstream of ERK, PKG II inhibited the phosphorylation of MEK1/2, the phosphorylation/activation of Raf-1, the activation of Ras, and the binding between adaptor protein Grb2 and GTP exchange factor Sos1 induced by EGF. Of note, PKG II inhibited the tyrosine phosphorylation of EGFR induced by EGF. Downstream of ERK, the EGF-induced nuclear translocation of phospho-ERK was also inhibited by PKG II. The results suggest that PKG II inhibits the proliferation of gastric cancer cells through blocking EGF-triggered MAPK signal transduction and the key blocking point is the tyrosine phosphorylation of the EGF receptor.

Cortical traumatic brain injury (TBI) is a major cause of cognitive impairment accompanied by motor and behavioral deficits, and there is no effective treatment strategy in the clinic. Cell transplantation is a promising therapeutic strategy, and it is necessary to verify the survival and differentiation of cells after transplantation in large animal models like rhesus monkeys. In this study, we transplanted neural stem cells (NSCs) and simultaneously injected basic fibroblast growth factor/epidermal growth factor (bFGF/EGF) into the cortex (visual and sensory cortices) of rhesus monkeys with superficial TBI. The results showed that the transplanted NSCs did not enter the cerebrospinal fluid (CSF) and were confined to the transplantation site for at least one year. The transplanted NSCs differentiated into mature neurons that formed synaptic connections with host neurons, but glial scar formation between the graft and the host tissue did not occur. This study is the first to explore the repairing effect of transplanting NSCs into the superficial cerebral cortex of rhesus monkeys after TBI, and the results show the ability of NSCs to survive long-term and differentiate into neurons, demonstrating the potential of NSC transplantation for cortical TBI. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies revealed that type II cGMP‐dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR) which is a widely investigated RTK. PDGFR belongs to family of receptor tyrosine kinases (RTKs) too. However, the effect of PKG II on PDGFR activation is not clear yet. This study investigated potential regulatory effect of PKG II on activation of PDGFRβ and the downstream signaling transductions in gastric cancer. The results from CCK8 assay and Transwell assay indicated that PDGF‐BB induced cell proliferation and migration. Activated PKG II reversed the above variations caused by PDGF‐BB. Immunoprecipitation and Western blotting results showed that PKG II combined with PDGFRβ and phosphorylated this receptor, and thereby inhibited PDGF‐BB induced activation of PDGFRβ, and MAPK/ERK and PI3K/Akt mediated signal transduction pathways. Based on the prediction by phosphorylation site software, Ser643 and Ser712 were mutated to alanine respectively which prevented phosphorylation at these sites. Mutation at Ser712 abolished the inhibitory function of PKG II on PDGFRβ activation but mutation of Ser643 had no such an effect, indicating that Ser712 was PKG II‐specific phosphorylating site of PDGFRβ. In conclusion, PKG II inhibited PDGFRβ activation in gastric cancer via phosphorylating Ser712 of this RTK. [ABSTRACT FROM AUTHOR]

PKG II, cGMP-dependent protein kinase type II; EGF, epidermal growth factor; Ad-LacZ, adenoviral vector containing the -galactosidase gene; Ad-PKG II, adenoviral vector containing the PKG II gene; cGMP, cyclic guanosine monophosphate. PKG II, cGMP-dependent protein kinase type II; Ad-LacZ, adenoviral vector containing the -galactosidase gene; Ad-PKG II, adenoviral vector containing the PKG II gene; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP; cGMP, cyclic guanosine monophosphate; EGF, epidermal growth factor. [Extracted from the article]

Type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG II) is a membrane-anchored enzyme expressed mainly in the intestinal mucosa and the brain, and is associated with various physiological or pathological processes. Upregulation of PKG II is known to induce apoptosis and inhibit proliferation and metastasis of cancer cells. The inhibitory effect of PKG II has been shown to be dependent on the inhibition of the activation of epidermal growth factor receptor (EGFR) and blockade of EGFR downstream signal transduction in vitro. However, it remains unclear whether similar phenomena/mechanisms exist in vivo and whether these effects are independent of cGMP or cGMP analogues. In the present work, nude mice with transplanted orthotopic tumours were infected with adenovirus encoding cDNA of constitutively active PKG II mutant (Ad-a-PKG II) and the effect of constitutively active PKG II (a-PKG II) on tumour development was detected. The results showed that a-PKG II effectively ameliorated gastric tumour development through delaying the growth, inducing the apoptosis, and inhibiting the metastasis and angiogenesis. The effect was related to blockade of EGFR activation and abrogation of the downstream signalling cascades. These findings provide novel insight which will benefit the development of new cancer therapies. [ABSTRACT FROM AUTHOR]

Our previous study demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF) induced tyrosine phosphorylation/activation of the EGF receptor (EGFR). This paper was designed to investigate the mechanism of the inhibition of PKG II on EGFR activation. Gastric cancer cells HGC-27 and AGS were infected with an adenoviral vector encoding the cDNA of PKG II (Ad-PKG II) to overexpress PKG II and treated with 8-(4-chlorophenylthio) guanosine-3′,5′-cyclic monophosphate (8-pCPT-cGMP) to activate the kinase. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay were performed to detect the interaction between PKG II and EGFR. Western blotting, mass spectrometry (MS) and site mutagenesis were performed to detect the PKG II-specific phosphorylation site on EGFR. The results showed that in living COS-7 cells, which were infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was an interaction between PKG II and EGFR. The results also showed that PKG II caused threonine 669 (T669) phosphorylation of EGFR in HGC-27 and AGS cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then inhibited the activation of EGFR. When T669 of EGFR was mutated to alanine, the inhibitory effect of PKG II on the activation of EGFR was eradicated. These findings suggested a PKG II-specific phosphorylation site on EGFR, and might be beneficial to illuminate the anti-tumor role of PKG II. • Interaction between PKG II and EGFR was detected in living cells. • PKG II caused T669 phosphorylation of EGFR. • PKG II inhibited the activation of EGFR via phosphorylating its T669. [ABSTRACT FROM AUTHOR]