P21-activated Kinase Research Articles

The role of p21-activated kinase 4 in the progression of oral squamous cell carcinoma by targeting PI3K-AKT signaling pathway.

Oral squamous carcinoma (OSCC), the most common head and neck malignancy, has a strong propensity for malignant proliferation and metastasis, which will decrease the survival of patients. P21-activated kinase 4 (PAK4), a classical serine/threonine protein kinase with multiple cellular functions, has an essential role in cancer cell migration and invasion. Here, we elucidated the function and possible molecular mechanisms of the effect of PAK4 on the biological behaviors of OSCC. The expression of genes and protein was detected by real-time PCR and western blotting. We used oral squamous carcinoma cell lines, Tca8117, Cal 27, SCC 4, and SCC 9 for validation of our cell function data. Flow cytometry, 3D cultures, and clone formation assay were used to detect proliferation of cells. RNA sequencing and bioinformatic analysis was performed to determine the potential function of PAK4. Immunohistochemistry, western blotting and real-time PCR demonstrated that PAK4 expression was up-regulated in OSCC tissues. Overexpression of PAK4 promoted the proliferation, migration and invasion of OSCC cell lines. RNA sequencing (RNA-seq) for the transcriptome-wide analysis of differential gene expression followed by bioinformatic analysis was performed to determine the potential function of PAK4. Based on the KEGG enrichment analysis and GO analysis of differential expression genes (DEGs) we found that PAK4 promotes the cell-cycle machinery, which associated with 44 regulated genes, thereby promoting cancer cell differentiation. This study demonstrates that the PAK4 regulates the biological behaviors of OSCC by PI3K-AKT signaling pathway, and these findings might provide a novel strategy for OSCC treatment.

Diosmetin alleviates benzo[a]pyrene-exacerbated H1N1 influenza virus-induced acute lung injury and dysregulation of inflammation through modulation of the PPAR-γ-NF-κB/P38 MAPK signaling axis.

The severity of a viral respiratory illness was greatly exacerbated after exposure to a contaminant containing benzo[a]pyrene (B[a]P). Flavonoid-rich fruit intake has gained intense interest due to their health-promoting benefits for viral respiratory diseases, including influenza viruses. In our study, diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a naturally occurring hydroxylated methoxyflavone that is abundant in Citrus fruits, was explored for its effects on B[a]P-exacerbated H1N1 influenza virus-mediated inflammation and lung injury. Initially, in vivo results demonstrated that diosmetin protected against H1N1 virus-elicited acute lung injury. Simultaneously, H1N1 virus or B[a]P-stimulated A549 cells treated with diosmetin inhibited NF-κB and P-P38 activation, resulting in suppression of pro-inflammatory cytokines and apoptosis. Interestingly, diosmetin obviously promoted the expression of PPAR-γ as well as nuclear translocation of PPAR-γ, whereas, PPAR-γ inhibition by GW9662 weakened the inhibitory effects of diosmetin on H1N1 virus or B[a]P-mediated activation of NF-κB and P-P38, elevated expression of pro-inflammatory mediators as well as apoptosis. Furthermore, it was surprising to discover that mice exposed to both B[a]P and H1N1 viruses contributed to exacerbated acute lung injury, which were significantly ameliorated by diosmetin administration. In vitro studies showed that A549 cells with the combination of B[a]P and H1N1 virus augmented NF-κB and P-P38 activation, accompanied by higher levels of pro-inflammatory mediators and apoptosis, all of which were also significantly reduced by diosmetin treatment. Repressing PPAR-γ abrogated the inhibitory effects of diosmetin on B[a]P-exacerbated H1N1 virus-mediated NF-κB and P-P38 activation, inflammation, and apoptosis in A549 cells. Our findings suggest that diosmetin protected against B[a]P-exacerbated H1N1 virus-mediated lung injury by suppressing the exacerbation of NF-κB and P38 kinase activation in a PPAR-γ-dependent manner, suggesting potential benefits for B[a]P-exacerbated influenza-related illness therapeutics.

Abstract 1316: Phosphorylation of Ribosomal Proteins by p21-Activated Protein Kinase 2 (PAK2) is a novel mechanism to inhibit mRNA translation under oxidative stress

Abstract 1316: Phosphorylation of Ribosomal Proteins by p21-Activated Protein Kinase 2 (PAK2) is a novel mechanism to inhibit mRNA translation under oxidative stress

The Role of MAP Kinases in the Induced Histone H2AX Phosphorylation in Transformed Cells

Previously, we have shown that inhibitors of histone deacetylases (HDIs) do not induce DNA double strand breaks (DNA DSBs). However, like genotoxic agents, HDIs initiate the accumulation of phosphorylated histone H2AX (γH2AX), which is a DNA DSB marker. HDIs can also reduce the efficiency of repair of DNA damaged by genotoxic effects in transformed cells. The aim of this work was to identify the signaling pathways leading to the accumulation of γH2AX under the HDIs treatment in transformed cells. There was considered the role of the MAPK family kinases in phosphorylation of histone H2AX as well as inhibition of DNA repair induced with HDI sodium butyrate (NaBut). It was shown that the accumulation of γH2AX under the NaBut treatment is accompanied by a decrease of the ERK and PKB/Akt kinases phosphorylation level in transformed cells. The activating phosphorylation of p38 kinase increases under the NaBut treatment, causing Wip1 phosphatase accumulation, which may be one of the reasons for the DNA repair inhibition. Suppression of p38 kinase activity abolishes the NaBut-induced inhibition of repair efficiency. The data obtained suggest the role of the p38/Wip1 pathway in the HDIs-induced decrease in repair efficiency in transformed cells.

Polarized contact behavior in directionally migrating Xenopus gastrula mesendoderm.

The control of cell-cell adhesion and detachment is essential for collective migration and cell rearrangement. Here, we have used the contact behavior of Xenopus gastrula mesoderm explants migrating directionally on ectoderm conditioned substratum to study the regulation of active cell-cell detachment. When colliding laterally, explants repelled each other, whereas they fused front-to-back when aligned in the direction of migration. For this mesoderm polarization by the substratum, we identified three control modules. First, PDGF-A signaling normally suppresses contact-induced collapse of lamellipodia in a polarized manner. Disruption of PDGF-A function, or of Xwnt6, decreased the polarization of explant contact behavior. Second, the Wnt receptor Xfz7 acted upstream of the kinase Pak1 to control explant fusion independently of PDGF-A-promoted lamellipodia stability. Third, ephrinB1 acted with Dishevelled (Dvl) in front-to-back explant fusion. The second and third modules have been identified previously as regulators of tissue separation at the ectoderm-mesoderm boundary. On non-polarizing, fibronectin-coated substratum, they controlled repulsion between explants in the same way as between tissues during boundary formation. However, explant repulsion/fusion responses were reversed on conditioned substratum by the endogenous guidance cues that also control oriented contact inhibition of lamellipodia. Together, control modules and substratum-bound guidance cues combine preferential front-back adhesion and diminished lateral adhesion of cells to promote collective directional mesoderm migration.

Involvement of the PRL-PAK1 Pathway in Cancer Cell Migration.

Prolactin (PRL) is a polypeptide hormone synthesized in the lactotrophs of the adenohypophysis and in extrahypophyseal glands (such as the prostate and breasts) where it promotes their development. PRL is also involved in cancer development in these glands. It has been shown to stimulate cancer cell migration, suggesting its possible involvement in metastasis, in which cell migration plays an essential role. However, the role of PRL in cell migration is still unclear. Moreover, the intracellular mechanisms activated by PRL to carry out cell migration are less well understood. PRL exerts its effects via the PRL receptor (PRLR), which leads intracellularly to phosphorylation of Janus protein kinase 2 (JAK2), which in turn phosphorylates p21-activated protein kinase (PAK1), leading to an increase in cell migration. Although several studies have described the involvement of the PRL-PAK1 pathway in breast cancer cell migration, the molecular mechanisms have not been fully elucidated and there is no integration of these into signaling pathways. This study was conducted based on literature search of review articles and original research in the PubMed database, using the following keywords: PRL, cell migration, PRL and cell migration, PAK1 and signaling pathways. The aim of this review article was to describe the major signaling pathways controlled by PRL-PAK1 and propose a comprehensive model of the signaling pathways associated with PRL-PAK1.

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor

AbstractIn response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel−Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Inka2 expression in smooth muscle cells and its involvement in cell migration

The cell motility of smooth muscle cells (SMCs) is essential for vascular and internal organ development and tissue regeneration in response to damage. Cell migration requires dynamic changes in the actin-cytoskeleton via the p-21 activated kinase (Pak)-Cofilin signaling cascade, which is the central axis of the actin filaments. We previously identified that the Inka2 gene was preferentially expressed in the central nervous system (CNS) and revealed that Inka2 directly binds Pak4 to suppress its kinase activity, thereby regulating actin de-polymerization in dendritic spine formation of the forebrain neurons. However, its physiological significance outside the CNS remains unclear. Here we determined the Inka2 expression profile in various organs using in situ hybridization analysis and lacZ staining on Inka2flox/+ mice. Robust Inka2 expression was consistently detected in the SMCs of many peripheral organs, including the arteries, esophagus, stomach, intestine, and bladder. The scratch assay was used on primary cultured SMCs and revealed that Inka2−/− SMC exhibits accelerated cell migration ability without a change in the cell proliferation rate. Inka2−/− SMCs displayed Cofilin activation/phosphorylation, a downstream molecule of Pak4 signal cascade. These results suggest that Inka2 regulates SMC motility through modulating actin reorganization as the endogenous inhibitor of Pak4.

The P21-Activated Kinase 1 and 2 As Potential Therapeutic Targets for the Management of Cardiovascular Disease

Review The P21-Activated Kinase 1 and 2 As Potential Therapeutic Targets for the Management of Cardiovascular Disease Honglin Xu, Dingwei Wang, Chiara Ramponi, Xin Wang, and Hongyuan Zhang * Michael Smith building, Division of Cardiovascular Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK. * Correspondence: hongyuan.zhang-3@postgrad.manchester.ac.uk Received: 20 October 2022 Accepted: 16 November 2022 Published: 21 December 2022 Abstract: Group I p21-activated kinases (Paks) are members of the serine/threonine protein kinase family. Paks are encoded by three genes (Pak 1‒3) and are involved in the regulation of various biological processes. Pak1 and Pak2 are key members, sharing 91% sequence identity in their kinase domains. Recent studies have shown that Pak1/2 protect the heart from various types of stresses. Activated Pak1/2 participate in the maintenance of cellular homeostasis and metabolism, thus enhancing the adaptation and resilience of cardiomyocytes to stress. The structure, activation and function of Pak1/2 as well as their protective roles against the occurrence of cardiovascular disease are described in this review. The values of Pak1/2 as therapeutic targets are also discussed.

Loss-of-Function of p21-Activated Kinase 2 Links BMP Signaling to Neural Tube Patterning Defects.

Closure of the neural tube represents a highly complex and coordinated process, the failure of which constitutes common birth defects. The serine/threonine kinase p21-activated kinase 2 (PAK2) is a critical regulator of cytoskeleton dynamics; however, its role in the neurulation and pathogenesis of neural tube defects (NTDs) remains unclear. Here, the results show that Pak2-/- mouse embryos fail to develop dorsolateral hinge points (DLHPs) and exhibit craniorachischisis, a severe phenotype of NTDs. Pak2 knockout activates BMP signaling that involves in vertebrate bone formation. Single-cell transcriptomes reveal abnormal differentiation trajectories and transcriptional events in Pak2-/- mouse embryos during neural tube development. Two nonsynonymous and one recurrent splice-site mutations in the PAK2 gene are identified in five human NTD fetuses, which exhibit attenuated PAK2 expression and upregulated BMP signaling in the brain. Mechanistically, PAK2 regulates Smad9 phosphorylation to inhibit BMP signaling and ultimately induce DLHP formation. Depletion of pak2a in zebrafish induces defects in the neural tube, which are partially rescued by the overexpression of wild-type, but not mutant PAK2. The findings demonstrate the conserved role of PAK2 in neurulation in multiple vertebrate species, highlighting the molecular pathogenesis of PAK2 mutations in NTDs.

PAK4 inhibition improves PD1 blockade immunotherapy in prostate cancer by increasing immune infiltration

Antitumor immunity requires lymphocytes to localize to the tumor. Prostate cancers (PCs) are immunologically cold and tend to lack T-cell infiltration. Most advanced PCs are insensitive to PD1 blockade therapies. Using syngeneic RM1 prostate tumors, p21-activated kinase-4 (PAK4) knockdown (KD) and pharmacological inhibition was assessed in C57BL/6J mice treated with PD1 antibodies (αPD1). RNASeq was used to characterize the immune response in the tumor. Immunohistochemistry, flow cytometry, and in vivo blocking studies confirmed the role of cell surface proteins in the generation of immune responses. In The Cancer Genome Atlas, PAK4 expression was inversely correlated with immune cell infiltration. PAK4 expression was controlled by the androgen receptor and its pioneering factor, FOXA1. PAK4 KD increased CD8+ T-cell infiltration and expression of IFNγ response genes. PAK4 KD also upregulated angiogenesis and endothelial cell adhesion molecules in the tumor microenvironment, contributing to CD8+ lymphocyte recruitment. Pharmacological inhibition of PAK4 made PC more responsive to immunotherapy with αPD1. A decrease in PAK4 activity increases immune activation and vascularity, which increases CD8+ lymphocyte infiltration into the tumor. Therefore, targeting PAK4 may improve the response of human PC to immunotherapy.

Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

Pan-cancer analysis identifies the immunological and prognostic role of PAK4

AimsP21-activated kinase 4 (PAK4) belongs to the wider family of Serine/Threonine p21-activated kinases (PAKs) and functions as a hub for signaling pathways in cancer progression. Numerous studies have indicated the significance of PAK4 for tumorigenesis, but no systematic pan-cancer analysis has been performed. Main methodsThe current study aimed to investigate the prognostic and immunological functions of PAK4 through bioinformatic analysis of datasets from The Cancer Genome Atlas, UALCAN, GEPIA2, cBioPortal, TIMER2, and Human Protein Atlas. PAK4 expression was correlated with prognosis, DNA methylation, tumor mutational burden, microsatellite instability, and immune cell infiltration. Key findingsPAK4 was highly expressed in various cancers but showed decreased expression in colon adenocarcinoma, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, and thyroid carcinoma. PAK4 was found to have a positive or negative correlation with prognosis of different cancers. PAK4 expression was related to tumor mutational burden in 11 tumor types, and associated with microsatellite instability in 10 tumor types and was correlated with immune infiltration and immune checkpoint genes. SignificancePAK4 could be considered as a prognostic and immunotherapeutic marker for some types of malignant tumor.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

C-Phycocyanin and Lycium barbarum Polysaccharides Protect against Aspirin-Induced Inflammation and Apoptosis in Gastric RGM-1 Cells

Aspirin causes gastrotoxicity and damaged epithelial defense via cyclooxygenase inhibition. C-phycocyanin (CPC) and Lycium barbarum polysaccharides (LBP), an active ingredient of Spirulina platensis and wolfberry, respectively, exerted antioxidation, anti-inflammation, and/or immunoregulation. The actions of CPC and/or LBP on gastric damage induced by aspirin were explored in rat gastric mucosal RGM-1 cells. Gastric injury was performed by 21 mM aspirin for 3 h after the pretreatment of CPC and/or LBP (100-500 μg/mL) for 24 h in RGM-1 cells. Proinflammatory, anti-inflammatory, and apoptotic markers were examined by ELISA or gel electrophoresis and Western blotting. Cell viability and interleukin 10 (IL-10) were reduced by aspirin. Increased proinflammatory markers, caspase 3 activity, and Bax protein were observed in RGM-1 cells with aspirin treatment. Aspirin elevated nuclear factor-κB (NF-κB), extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) activation, while CPC and/or LBP increased IL-10, and attenuated proinflammatory markers, Bax protein, NF-κB, and the activation of ERK and JNK. Therefore, CPC and/or LBP possess anti-inflammation by restraining the activation of the ERK signaling pathway, and LBP decreases apoptosis by suppressing the JNK signaling pathway activation in gastric RGM-1 cells with aspirin-induced epithelial damage.

PP018 PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling

PP018 PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling

MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a prevalent illness in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) assist in ARDS lung injury and the inflammatory process through mechanisms that are unclear. This study investigated the role of AM-derived secretory autophagosomes (SAPs) in lung injury and microRNA (MiR)-199a-3p-regulated inflammation associated with ARDS in vitro and in a murine model. The ARDS model in mouse was established by intratracheal LPS lipopolysaccharide (LPS) injection. The agomirs or antagomirs of MiR-199a-3p were injected into the caudal vein to figure out whether MiR-199a-3p could influence ARDS inflammation and lung injury, whereas the mimics or inhibitors of MiR-199a-3p, siRNA of Rab8a, or PAK4 inhibitor were transfected or applied to RAW264.7 cells to evaluate the mechanism of SAP release. Culture supernatants of RAW264.7 cells treated with LPS or bronchoalveolar lavage fluid from mice were collected for the isolation of SAPs. We found that MiR-199a-3p was over-expressed in the lungs of ARDS mice. The MiR-199a-3p antagomir alleviated, whereas the MiR-199a-3p agomir exacerbated LPS-induced inflammation in mice by promoting AM-derived SAP secretion. In addition, MiR-199a-3p over-expression exacerbated LPS-induced ARDS via activating Rab8a, and Rab8a silencing significantly suppressed the promoting influence of the MiR-199a-3p mimic on SAP secretion. Furthermore, MiR-199a-3p mimic activated Rab8a by directly inhibiting PAK4 expression. The novel finding of this study is that MiR-199a-3p participated in the regulation of SAP secretion and the inflammatory process via targeting of PAK4/Rab8a, and is a potential therapeutic candidate for ARDS treatment.

Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis

ABSTRACT With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn’s disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune–microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.

The role of the miR-4306/PAK6 axis in degenerative nucleus pulposus cells in human intervertebral disc degeneration

Intervertebral disc degeneration (IDD), characterized by degenerative changes that occur in intervertebral discs due to aging or structural injury, is thought to be the most common cause of lower back pain. Recent studies have shown that microRNAs (miRNAs) have a critical role in the etiopathogenesis of IDD. In the current study, we aimed to determine the role of miRNAs in mediating the underlying mechanisms associated with IDD. First, differentially expressed miRNAs (DEmiRNAs) were identified using the GEO database, and subsequently confirmed by RT-qPCR and in situ hybridization. We found that miR-4306 expression was significantly decreased in human nucleus pulposus (NP) tissues compared with healthy controls, and was negatively correlated with the patients' Pfirrmann grade. To determine the mechanism by which miR-4306 was involved in IDD pathogenesis, we examined the effects of overexpressing or silencing miR-4306 on extracellular matrix (ECM) synthesis/degradation, proliferation, autophagy and apoptosis of human degenerated NP cells isolated from IDD patients. Next, we used dual-luciferase reporter assays to demonstrate that miR-4306 interacted with the 3′-untranslated regions of p21-activated kinase 6 (PAK6) mRNA, resulting in significant suppression of PAK6 expression. This effect was abolished by miR-4306 binding site mutations. Using miR-4306/PAK6 gain-of-function and loss-of-function studies in human degenerated NP cells, we demonstrated that miR-4306 promoted NP cell proliferation, ECM synthesis and autophagy, while inhibiting apoptosis and ECM degradation via PAK6. Thus, our findings indicate that miR-4306, acting via PAK6, has an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

Dienogest May Reduce Estradiol- and Inflammatory Cytokine-Induced Cell Viability and Proliferation and Inhibit the Pathogenesis of Endometriosis: A Cell Culture- and Mouse Model-Based Study.

Dienogest (DNG) is a therapeutic medication used in endometriosis treatment. Limited data are available regarding its mechanism of action on endometrial cells. Using in vivo and in vitro models, we investigated whether DNG treatment causes significant biological changes in human endometrial stromal cells (ESCs). The markers related to the pathogenesis of endometriosis in ESCs were evaluated using estradiol, tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-32, administered alone or in combination with DNG. Implanted endometrial tissues were compared between C57BL/6 mice that did or did not receive DNG treatment by using size measurements and immunohistochemistry. A significant decrease in cell viability, protein kinase B (AKT) phosphorylation, and the expression of p21-activated kinase 4 and vascular endothelial growth factor were observed in ESCs treated with estradiol plus DNG. Cell viability, AKT phosphorylation, and proliferating cell nuclear antigen (PCNA) expression also decreased significantly after TNF-α plus DNG treatment. Treatment with IL-1β or IL-32 plus DNG significantly decreased cell viability or PCNA expression, respectively. The size of the implanted endometrial tissue significantly decreased in mice treated with DNG, accompanied by decreased PCNA expression. Thus, DNG may reduce cell viability and proliferation induced by estradiol, TNF-α, IL-1β, and IL-32, and inhibit the endometriosis pathogenesis by decreasing PCNA expression.