P21-activated kinase 5 (PAK5) belongs to the PAK-II subfamily, which is an important regulator of cell survival, adhesion, and motility. However, the functions of PAK5 in endometriosis remain unclear. Here, PAK5 is strikingly upregulated in endometriosis. Furthermore, the knockdown of PAK5 or its inhibitor GNE 2861 blocks the development of endometriosis, which is equally demonstrated in PAK5-knockout mice. In addition, PAK5 promotes glycolysis by enhancing the protein stability of pyruvate kinase 2 (PKM2) in endometriotic cells, which is a key enzyme for glucose metabolism. Moreover, the phosphorylation of PKM2 at Ser519 by PAK5 mediates endometriosis cell proliferation and metastasis. Collectively, PAK5 plays an indispensable role in endometriosis. Our findings demonstrate that PAK5 is an important target for the treatment of endometriosis.
Endometriosis/genetics* ; Female ; Animals ; p21-Activated Kinases/genetics* ; Mice ; Phosphorylation ; Glycolysis ; Humans ; Thyroid Hormone-Binding Proteins ; Membrane Proteins/genetics* ; Carrier Proteins/genetics* ; Cell Proliferation ; Mice, Knockout ; Thyroid Hormones/metabolism* ; Pyruvate Kinase/genetics*

OBJECTIVE: To investigate the clinical phenotype and genetic basis of a child with epilepsy and global developmental delay. METHODS: A child with epilepsy and global developmental delay who had visited West China Second University Hospital, Sichuan University on April 1, 2021 was selected as the study subject. Clinical data of the child were reviewed. Genomic DNA was extracted from peripheral blood samples of the child and his parents. Whole exome sequencing (WES) was carried out for the child, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. A literature review was also carried out by searching databases such as Wanfang data knowledge service platform, China National Knowledge Infrastructure, PubMed, ClinVar and Embase to summarize the clinical phenotypes and genotypes of the affected children. RESULTS: The child was a 2-year-and-2-month-old male with epilepsy, global developmental delay and macrocephaly. Results of WES showed that the child has harbored a c.1427T>C variant of the PAK1 gene. Sanger sequencing confirmed that neither of his parents has carried the same variant. Only one similar case had been recorded by the dbSNP, OMIM, HGMD, and ClinVar databases. No frequency for this variant among Asian population was available in the ExAC, 1000 Genomes, and gnomAD databases. Prediction with IFT, PolyPhen-2, LRT, Mutation Taster, and FATHMM online software suggested that this variant is deleterious to the function of encoded protein. Based on the Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics (ACMG), the PAK1 gene c.1427T>C variant was determined to be likely pathogenic. CONCLUSION The PAK1 gene c.1427T>C variant probably underlay the epilepsy and global developmental delay in this child, which has provided a reference for the clinical diagnosis and genetic counseling in children with similar disorders.
Humans ; Male ; China ; Computational Biology ; Consensus ; Epilepsy/genetics* ; Genotype ; Mutation ; p21-Activated Kinases/genetics* ; Child, Preschool

OBJECTIVE: To explore the genetic etiology for a child with profound intellectual disabilities and obvious behavioral abnormalities. METHODS: A male child who had presented at the Zhongnan Hospital of Wuhan University on December 2, 2020 was selected as the study subject. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Short tandem repeat (STR) analysis was carried out to determine its parental origin. The splicing variant was also validated in vitro with a minigene assay. RESULTS: WES results revealed that the child had harbored a novel splicing variant of c.176-2A>G in the PAK3 gene, which was inherited from his mother. The results of minigene assay have confirmed aberrant splicing of exon 2. According to the guidelines from the American College of Medical Genetics and Genomics, it was classified as a pathogenic variant (PVS1+PM2_Supporting+PP3). CONCLUSION The novel splicing variant c.176-2A>G of the PAK3 gene probably underlay the disorder in this child. Above finding has expanded the variation spectrum of the PAK3 gene and provided a basis for genetic counseling and prenatal diagnosis for this family.
Child ; Female ; Humans ; Male ; Pregnancy ; Exons ; Intellectual Disability/genetics* ; Mothers ; Mutation ; p21-Activated Kinases/genetics* ; Parents ; RNA Splicing

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 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*

BACKGROUND/AIMS: 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells. METHODS: We used FRO thyroid cancer cells harboring the BRAF(V600E) mutation to examine the effect of AICAR on cell proliferation and cell survival. We also evaluated the involvement of mitogen-activated protein kinase (MAPK) pathways in this effect. RESULTS: We found that AICAR treatment promoted AMPK activation and suppressed cell proliferation and survival by inducing p21 accumulation and activating caspase-3. AICAR significantly induced activation of p38 MAPK, and pretreatment with SB203580, a specific inhibitor of the p38 MAPK pathway, partially but significantly rescued cell survival. Furthermore, small interfering RNA targeting AMPK-alpha1 abolished AICAR-induced activation of p38 MAPK, p21 accumulation, and activation of caspase-3. CONCLUSIONS: Our findings demonstrate that AMPK activation using AICAR inhibited cell proliferation and survival by activating p38 MAPK and proapoptotic molecules in FRO thyroid cancer cells. These results suggest that the AMPK and p38 MAPK signaling pathways may be useful therapeutic targets to treat thyroid cancer.
AMP-Activated Protein Kinases/genetics/metabolism ; Aminoimidazole Carboxamide/*analogs & derivatives/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Activators/pharmacology ; Humans ; Mutation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; RNA Interference ; Ribonucleotides/*pharmacology ; Signal Transduction/*drug effects ; Thyroid Neoplasms/*enzymology/genetics/pathology ; Time Factors ; Transfection ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

BACKGROUNDPeg-Interferon-α treatment is expensive and associated with considerable adverse effects, selection of patients with the highest probability of response is essential for clinical practice. The objective of this study was to assess the relationship between the gene polymorphisms of interleukin-28 (IL-28), p21-activated protein kinase 4 (PAK4) and the response to interferon treatment in chronic hepatitis B patients.

METHODSTwo hundred and forty interferon-naive treatment HBeAg seropositive chronic hepatitis B patients were enrolled in the present prospective nested case-control study. Peripheral blood samples were collected, including 92 with favorable response and 148 without response to the interferon treatment. Rs8099917, rs12980602, and rs9676717 SNP was genotyped using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

RESULTSIL-28 genotype was not associated with response to interferon treatment (OR for GT/GG vs. TT, 0.881 (95%CI 0.388 - 2.002); P = 0.762; OR for CT/CC vs. TT, 0.902 (95%CI 0.458 - 1.778); P = 0.766). Rs9676717 in PAK4 genotype was independently associated with the response (OR for CT/CC vs. TT, 0.524 (95%CI 0.310 - 0.888); P = 0.016). When adjusting for age, gender, smoking, drinking, levels of hepatitis B virus DNA, and alanine aminotransferase (ALT), rs9676717 genotype TT appeared to be associated with a higher probability of response for interferon treatment (OR, 0.155 (95%CI 0.034 - 0.700); P = 0.015).

CONCLUSIONGenotype TT for rs9676717 in PAK4 gene and no drinking may be predictive of the interferon-a treatment success.

Adult ; Case-Control Studies ; Female ; Genotype ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Interferon-alpha ; therapeutic use ; Interleukins ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Prospective Studies ; p21-Activated Kinases ; genetics

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

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

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