The serine/threonine p21-activated kinases (PAKs), as main effectors of the Rho GTPases Cdc42 and Rac, represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity. PAKs show wide expression in the brain, but they differ in specific cell types, brain regions, and developmental stages. PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines. PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders. Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment, comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases, which may also represent potential therapeutic targets of these diseases.
Animals ; Cytoskeleton/genetics* ; Humans ; Nervous System Diseases/genetics* ; Neurons/enzymology* ; Signal Transduction ; p21-Activated Kinases/metabolism*

Animals ; Gene Expression ; Mice ; Mice, Inbred Strains ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; genetics ; metabolism ; p21-Activated Kinases ; genetics

OBJECTIVETo study the role of p21-activated kinase-4 (PAK4) in the occurrence, progression and metastasis of breast cancer.

METHODPAK4 expression was detected in 35 cases of normal breast, 22 breast cystic hyperplasia, 28 breast adenofibroma, 37 breast cancer (including 7 non-invasive cancer, 9 early invasive cancer and 21 invasive cancer) and 13 metastatic breast cancer tissues using immunohistochemistry for a comparison of PAK4 expression and distribution.

RESULTSPAK4 was expressed mainly in the cytoplasm of the cancer cells, occasionally in the cell nuclei, and virtually not expressed in the matrix surrounding the breast cells. PAK4 positivity rates increased in the order of normal breast tissues, benign changes (including breast cystic hyperplasea and breast adenoma), breast cancer and metastatic cancer tissues; in the cancer tissues, the positivity rates increased in the order of non-invasive breast tumor, early invasive tumor and invasive tumor tissues.

CONCLUSIONPAK4 is closely correlated to the progression and metastasis of breast cancer and may become a new diagnostic and therapeutic target of breast cancer.

Adult ; Aged ; Biomarkers, Tumor ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Female ; Humans ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Young Adult ; p21-Activated Kinases ; genetics ; metabolism

OBJECTIVETo investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene.

METHODSHuman colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting.

RESULTSSW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h.

CONCLUSIONEGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.

Blotting, Western ; Catechin ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; p21-Activated Kinases ; metabolism

BACKGROUND AND OBJECTIVEMicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells.

METHODSWe detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion.

RESULTSSimilar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05).

CONCLUSIONSMembers of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; MicroRNAs ; genetics ; metabolism ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Phosphorylation ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; cdc42 GTP-Binding Protein ; metabolism ; p21-Activated Kinases ; metabolism

BACKGROUND: Cell competition is an important feature in pancreatic cancer (PC) progression, but the underlying mechanism remains elusive. This study aims to explore the role of exosomes derived from normal pancreatic ductal epithelial cells involved in PC progression. METHODS: PC cells and pancreatic stellate cells (PSCs) were treated with exosomes isolated from pancreatic ductal epithelial cells. Cell proliferation was assessed by CCK8 assays. Cell migration and invasion were assessed by Transwell assays. PC and matched adjacent non-tumor tissue specimens were obtained from 46 patients pathologically diagnosed with PC at Peking University First Hospital from 2013 to 2017. Tissue miR-485-3p and p21-activated kinase-1 (PAK1) expression was examined by real-time polymerase chain reaction (RT-PCR), and the relationship of the two was analyzed using Pearman's product-moment correlation. The clinical significance of miR-485-3p was analyzed using the Chi-square test, Wilcoxon rank-sum test, and Fisher exact probability, respectively. The binding of miR-485-3p to PAK1 5'-untranslated region (5'-UTR) was examined by luciferase assay. PC cells were xenografted into nude mice as a PC metastasis model. RESULTS: Exosomes from pancreatic ductal epithelial cells suppressed PC cell migration and invasion as well as the secretion and migration of PSCs. MiR-485-3p was enriched in the exosomes of pancreatic ductal epithelial cells but deficient in those of PC cells and PSCs, in accordance with the lower level in PSCs and PC cells than that in pancreatic ductal cells. And the mature miR-485-3p could be delivered into these cells by the exosomes secreted by normal pancreatic duct cells, to inhibit PC cell migration and invasion. Clinical data analysis showed that miR-485-3p was significantly decreased in PC tissues (P < 0.05) and was negatively associated with lymphovascular invasion (P = 0.044). As a direct target of miR-485-3p, PAK1 was found to exert an inhibitory effect on PC cells, and there was a significantly negative correlation between the expression levels of miR-485-3p and PAK1 (r = -0.6525, P < 0.0001) in PC tissues. Moreover, miR-485-3p could suppress PC metastasis in vivo by targeting p21-activated kinase-1. CONCLUSIONS Exosomal miR-485-3p delivered by normal pancreatic ductal epithelial cells into PC cells inhibits PC metastasis by directly targeting PAK1. The restoration of miR-485-3p by exosomes or some other vehicle might be a novel approach for PC treatment.
Animals ; Mice ; MicroRNAs/metabolism* ; Mice, Nude ; p21-Activated Kinases/metabolism* ; Cell Line, Tumor ; Pancreatic Neoplasms/genetics* ; Epithelial Cells/metabolism* ; Pancreatic Ducts/pathology* ; Cell Proliferation ; Cell Movement ; Gene Expression Regulation, Neoplastic

The integrity of blood vessels controls vascular permeability and extravasation of blood cells, across the endothelium. Thus, the impairment of endothelial integrity leads to hemorrhage, edema, and inflammatory infiltration. However, the molecular mechanism underlying vascular integrity has not been fully understood. Here, we demonstrate an essential role for A-kinase anchoring protein 12 (AKAP12) in the maintenance of endothelial integrity during vascular development. Zebrafish embryos depleted of akap12 (akap12 morphants) exhibited severe hemorrhages. In vivo time-lapse analyses suggested that disorganized interendothelial cell-cell adhesions in akap12 morphants might be the cause of hemorrhage. To clarify the molecular mechanism by which the cell-cell adhesions are impaired, we examined the cell-cell adhesion molecules and their regulators using cultured endothelial cells. The expression of PAK2, an actin cytoskeletal regulator, and AF6, a connector of intercellular adhesion molecules and actin cytoskeleton, was reduced in AKAP12-depleted cells. Depletion of either PAK2 or AF6 phenocopied AKAP12-depleted cells, suggesting the reduction of PAK2 and AF6 results in the loosening of intercellular junctions. Consistent with this, overexpression of PAK2 and AF6 rescued the abnormal hemorrhage in akap12 morphants. We conclude that AKAP12 is essential for integrity of endothelium by maintaining the expression of PAK2 and AF6 during vascular development.
A Kinase Anchor Proteins/*genetics/metabolism ; Animals ; Blood Vessels/abnormalities/*embryology/metabolism ; Cell Cycle Proteins/genetics/metabolism ; Down-Regulation ; Embryo, Nonmammalian/abnormalities/*blood supply/embryology/metabolism ; Gene Deletion ; *Gene Expression Regulation, Developmental ; Hemorrhage/*embryology/genetics/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Intercellular Junctions/genetics/metabolism/ultrastructure ; Kinesin/genetics/metabolism ; Myosins/genetics/metabolism ; Zebrafish/*embryology/genetics ; p21-Activated Kinases/genetics/metabolism

Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.
1-Phosphatidylinositol 3-Kinase/metabolism ; Angiotensin II/*metabolism ; Animals ; *Cell Movement ; Cells, Cultured ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Muscle, Smooth, Vascular/cytology ; Myocytes, Smooth Muscle/*cytology ; NADPH Oxidase/metabolism ; Protein Kinase C/metabolism ; RNA, Small Interfering/genetics ; Rats ; Rats, Sprague-Dawley ; p21-Activated Kinases/metabolism ; rac1 GTP-Binding Protein/metabolism ; src-Family Kinases/metabolism

OBJECTIVETo investigate the significance and mechanisms of overexpression of p21-activated kinase 1 gene (PAK1) in epithelial ovarian neoplasms.

METHODSImmunohistochemistry, fluorescence in situ hybridization and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling methods were used to examine the protein expression and amplification of PAK1 and cell apoptosis in 30 benign ovarian adenomas, 20 borderline tumors and 80 ovarian carcinomas by tissue microarray.

RESULTSIn immunohistochemistry study, overexpression of PAK1 protein was observed in 7 (25.9%) informative benign ovarian adenomas, 7 (36.8%) borderline tumors and 53 (68.8%) ovarian carcinomas. A significant inverse correlation of PAK1 overexpression and cell apoptosis was observed in these epithelial ovarian neoplasm cohorts (P = 0.002). In addition, 27/31 (87.1%) poorly differentiated (G3) carcinomas showed overexpression of PAK1, the frequency was significantly higher than that in tumors of G1 - G2 (26/46, 56.5% , P =0.01). In fluorescence in situ hybridization study, only 2 (4.7%) informative ovarian carcinomas showed amplification of PAK1 gene. None of the borderline and benign ovarian tumors showed PAK1 amplification.

CONCLUSIONOverexpression of PAK1 protein may be involved in the tumorigenesis of epithelial ovarian neoplasms and it is associated closely with the malignant histological phenotype of ovarian carcinomas. Mechanism other than gene amplification of PAK1 may play a more important role in the regulation of protein expression of PAK1 in ovarian tumors.

Adenoma ; genetics ; metabolism ; pathology ; Apoptosis ; Cystadenocarcinoma, Mucinous ; genetics ; metabolism ; pathology ; Cystadenocarcinoma, Serous ; genetics ; metabolism ; pathology ; Female ; Gene Amplification ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; In Situ Nick-End Labeling ; Middle Aged ; Neoplasm Staging ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; p21-Activated Kinases ; genetics ; metabolism

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21(Cip/WAF1) activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21(Cip/WAF1) short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.
Animals ; Arginine ; Cell Dedifferentiation ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Elongation Factor 2 Kinase/*metabolism ; Fibroblast Growth Factor 2/*metabolism ; Fibroblasts/*metabolism/pathology ; Flavonoids/pharmacology ; MAP Kinase Signaling System/drug effects/genetics ; Methylation ; Mice ; Mitogen-Activated Protein Kinases/antagonists & inhibitors ; Myofibroblasts/pathology ; NIH 3T3 Cells ; Protein Methyltransferases/*metabolism ; Protein-Arginine N-Methyltransferases/*metabolism ; RNA, Small Interfering/genetics