Effect Of PKGⅡ On LPA-Induced Migration And Activation Of Rhoa And Rac1 In Gastric Cancer Cells

Objective:To detect the expression of type Ⅱ cGMP-dependent protein kinase (PKG Ⅱ) in cancer cell lines and gastric cancer tissues. To study the effect of PKG Ⅱ on LPA-induced migration in gastric cancer cells. To identify the effect of PKG Ⅱ on LPA-induced activation of migration-related small GTPase RhoA and Racl.Methods:(1) To detect the expression of PKG Ⅱ in gastric cancer cell lines, breast cancer cell lines, kidney cancer cell lines and corresponding epithelial cell lines by RT-PCR. To detect the expression of PKG Ⅱ in gastric cancer tissues by qPCR. Wilcoxon Signed Ranks Test was used to analyze the difference between the gastric cancer tissue and corresponding adjacent normal tissue. Mann-Whitney U test or Kruskal-Wallish test was applied to analyze the correlation between expression of PKG Ⅱ and parameters of clinical pathology.(2) The gastric cancer cell lines AGS and HGC-27 were infected by adenoviral construct encoding PKG Ⅱ cDNA (Ad-PKG Ⅱ) to increase the expression of PKG Ⅱ. 8-pCPT-cGMP was used to activate the PKG Ⅱ specifically. LPA was applied to induce the migration activity of the cells. To analyze the migration of gastric cancer cells by Transwell migration assay. To detect the stress fibers in the gastric cancer cells by fluorescent staining. Pull-down assay and Western blotting were used to detect the activation of RhoA and Racl. Western blotting was applied to investigate the activation of key molecules in MAPK/ERK and PI3K/Akt pathways. Immunoprecipatation (IP) and Western blotting were used to detect the tyrosine phosphorylation of EGFR by PKG Ⅱ.(3) AGS and HGC-27 cells were respectively transfected by the plasmids of pFlag-RhoA, pFlag-RhoA188A or pFlag-Racl. Then the cells were infected by Ad-PKG Ⅱto increase PKG Ⅱ expression and were stimulated by 8-pCPT-cGMP to activate PKG Ⅱ. IP and Western blotting were used to analyze the serine and threonine phosphorylation of RhoA and Racl caused by PKG Ⅱ.(4) AGS cells were infected by Ad-PKG Ⅱ to increase PKG Ⅱ expression and PKG Ⅱ activity was increased by 8-pCPT-cGMP. Co-IP assay was used to detect the binding between PKG Ⅱ and RhoA. The plasmids pBiFC-RhoA-VC and pBiFC-PKG Ⅱ-VN were constructed and used to co-transfect COS-7 cells. The binding between PKG Ⅱ and RhoA in live cells was detected by bimolecular fluorescence complementation assay (BiFC). The prokaryotic expression plasmids of RhoA and PKG Ⅱ with different domain were constructed, and then were used to transform E.coli to express the recombinant proteins. Pull-down assay and Western blotting were applied to detect the binding between PKG Ⅱ and RhoA deletion mutants or RhoA and PKG Ⅱ deletion mutants.(5) AGS and HGC-27 cells were treated with inhibitors of MAPK/ERK pathway, PI3K/Akt pathway and PLC/IP3/DAG pathway. Pull-down assay and Western blotting were applied to analyze the activation of RhoA and Rac1.(6) AGS and HGC-27 cells were treated by EGFR inhibitor and then LPA. Pull-down assay and Western blotting were used to analyze the activation of Racl and RhoA. Western blotting was applied to detect the activation of key molecules in MAPK/ERK and PI3K/Akt pathways.Results:(1) Among human gastric mucosal, mammary gland and embryonic kidney epithelial cell, the human gastric mucosal epithelial cell line GES-1 had the highest level of PKG Ⅱ. Among the above 3 epithelial cell lines from different tissues and corresponding cancer cell lines, the most obvious difference of PKG Ⅱ expression was between gastric mucosal epithelial cell line and gastric cancer cell lines. In 49 cases out of 74 cases (66.2%), the expression of PKG Ⅱ in cancer tissues was lower than that in adjacent normal tissues (P<0.05). The change of PKG Ⅱexpression was not related with the clinical pathology parameters significantly.(2) Activated endogenous PKG Ⅱ inhibited LPA-induced migration of gastric mucosal epithelial cell line GES-1. Activated exogenous PKG Ⅱ inhibited LPA-induced increase of migration and cytoskeleton development of gastric cancer cell lines AGS and HGC-27.(3) Activated exogenous PKG Ⅱ inhibited LPA-induced activation of RhoA. PKG Ⅱ phosphorylated RhoA on serine/threonine residue. Serine 188 was the specific site of RhoA phosphorylated by PKG Ⅱ. PKG Ⅱ bound with RhoA, and the amino-terminus of PKG Ⅱ and RhoA were critical for the binding.(4) Activated exogenous PKG Ⅱ inhibited LPA-induced activation of Racl, but didn’t phosphorylate Racl significantly. PKG Ⅱ might decrease LPA-induced Rac1 activation by blocking LPA-induced activation of EGFR and consequently inhibiting the LPA-induced signal transduction of MAPK/ERK and PI3K/Akt pathways.Conclusions:The expression of PKG Ⅱ was lower in gastric cancer cells than that in human gastric mucosal epithelial cells, and lower in gastric cancer tissue than that in adjacent normal tissue. After being activated, endogenous and exogenous PKG Ⅱ inhibited LPA-induced migration in gastric mucosal epithelial cells and gastric cancer cells, respectively. PKG Ⅱ decreased the LPA-induced activation of RhoA and Rac1. PKG Ⅱ bound with RhoA and phosphorylated its 188 serine residue. PKG Ⅱ attenuated the LPA-induced activation of Racl by inhibiting the activation of EGFR and the downstream signaling of MAPK-ERK and PI3K-Akt pathways.