FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane.

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4A/D stability. Proteomic analysis reveals that FN stimulation induces phosphorylation at S143 of Arl4A and at S144 of Arl4D. We identify Pak1 as the responsible kinase for these phosphorylations. Moreover, these phosphorylations promote the chaperone protein HYPK to bind Arl4A/D, which stabilizes their recruitment to the plasma membrane to promote cell migration. These findings not only advance a major mechanistic understanding of how Arl4 proteins act in cell migration but also achieve a fundamental understanding of how these small GTPases are modulated by revealing that protein stability, rather than the GTPase cycle, acts as a key regulatory mechanism.

Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance.

Axon pathfinding is an essential step in neuronal network formation. Shootin1a is a clutch-linker molecule that is mechanically involved in axon outgrowth and guidance. It was previously shown that concentration gradients of axon guidance molecule netrin-1 in the extracellular environment elicit asymmetrically localized Pak1 kinase-mediated phosphorylation of shootin1a within axonal growth cones, which is higher on the netrin-1 source side. This asymmetric phosphorylation promotes shootin1a-mediated local actin-adhesion coupling within growth cones, thereby generating directional forces for turning the growth cone toward the netrin-1 source. However, how the spatial differences in netrin-1 concentration are transduced into the asymmetrically localized signaling within growth cones remains unclear. Moreover, the protein phosphatases that dephosphorylate shootin1a remain unidentified. Here, we report that protein phosphatase-1 (PP1) dephosphorylates shootin1a in growth cones. We found that PP1 overexpression abolished the netrin-1-induced asymmetric localization of phosphorylated shootin1a as well as axon turning. In addition, we show PP1 inhibition reversed the asymmetrically localized shootin1a phosphorylation within growth cones under netrin-1 gradient, thereby changing the netrin-1-induced growth cone turning from attraction to repulsion. These data indicate that PP1-mediated shootin1a dephosphorylation plays a key role in organizing asymmetrically localized phosphorylated shootin1a within growth cones, which regulates netrin-1-induced axon guidance.

Targeting endothelial PDGFR-ß facilitates angiogenesis-associated bone formation through the PAK1/NICD axis.

The intricate orchestration of osteoporosis (OP) pathogenesis remains elusive. Mounting evidence suggests that angiogenesis-driven osteogenesis serves as a crucial foundation for maintaining bone homeostasis. This study aimed to explore the potential of the endothelial platelet-derived growth factor receptor-ß (PDGFR-ß) in mitigating bone loss through its facilitation of H-type vessel formation. Our findings demonstrate that the expression level of endothelial PDGFR-ß is reduced in samples obtained from individuals suffering from OP, as well as in ovariectomy mice. Depletion of PDGFR-ß in endothelial cells ameliorates angiogenesis-mediated bone formation in mice. The regulatory influence of endothelial PDGFR-ß on H-type vessels is mediated through the PDGFRß-P21-activated kinase 1-Notch1 intracellular domain signaling cascade. In particular, the endothelium-specific enhancement of PDGFR-ß facilitates H-type vessels and their associated bone formation in OP. Hence, the strategic targeting of endothelial PDGFR-ß emerges as a promising therapeutic approach for the management of OP in the near future.

Antagonistic actions of PAK1 and NF2/Merlin drive myelin membrane expansion in oligodendrocytes.

In the central nervous system, the formation of myelin by oligodendrocytes (OLs) relies on the switch from the polymerization of the actin cytoskeleton to its depolymerization. The molecular mechanisms that trigger this switch have yet to be elucidated. Here, we identified P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. Our results demonstrate that PAK1 accumulates in OLs in a kinase-inhibited form, triggering actin disassembly and, consequently, myelin membrane expansion. Remarkably, proteomic analysis of PAK1 binding partners enabled the identification of NF2/Merlin as its endogenous inhibitor. Our findings indicate that Nf2 knockdown in OLs results in PAK1 activation, actin polymerization, and a reduction in OL myelin membrane expansion. This effect is rescued by treatment with a PAK1 inhibitor. We also provide evidence that the specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that the antagonistic actions of PAK1 and NF2/Merlin on the actin cytoskeleton of the OLs are critical for proper myelin formation. These findings have broad mechanistic and therapeutic implications in demyelinating diseases and neurodevelopmental disorders.

Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

Advanced glycation end products promote intervertebral disc degeneration by transactivation of matrix metallopeptidase genes.

OBJECTIVE: Examine the mechanism by which advanced glycation end products (AGEs) induce intervertebral disc degeneration (IDD) in C57BL/6J mice. METHODS: Matrix metallopeptidase (MMP) gene mRNA levels were assessed using RT-qPCR. Immunoprecipitation and co-immunoprecipitation were performed to identify the transcriptional complex regulating MMP expression due to AGEs. The preventive effects of inhibitors targeting this complex were tested in mice on high AGE diets. RESULTS: IDD and AGE accumulation were evident in mice on high-AGE diets (HAGEs), persisting across dietary shifts but absent in mice exclusively on low-AGE diets. Molecularly, HAGEs activated p21-activated kinase 1 (PAK1), prompting peroxisome proliferator-activated receptor gamma coactivator-related protein 1 (PPRC1) phosphorylation. Ubiquitin-specific protease 12 (USP12) interacted with the phosphorylated PPRC1 (pPPRC1), safeguarding it from proteasomal degradation. This pPPRC1, in collaboration with two histone acetyltransferases p300/CREB-binding protein (CBP) and a transcription factor activator protein 1(AP1), enhanced the expression of 12 MMP genes (MMP1a/1b/3/7/9/10/12/13/16/19/23/28). In vitro AGE exposure on nucleus pulposus and annulus fibrosus cells replicated this gene activation pattern, driven by the PAK1/pPPRC1-p300/CBP-AP1 pathway. The application of PAK1, p300, and AP1 inhibitors reduced pPPRC1-p300/CBP-AP1 binding to MMP promoters, diminishing their expression. These inhibitors effectively thwarted IDD in HAGE mice. CONCLUSION: Our results revealed that HAGEs instigate IDD via the PAK1/pPPRC1-p300/CBP-AP1 signaling pathway. This insight can guide therapeutic strategies to slow IDD progression in prediabetic/diabetic patients.

LncRNA MALAT1 facilitates BM-MSCs differentiation into endothelial cells and ameliorates erectile dysfunction via the miR-206/CDC42/PAK1/paxillin signalling axis.

BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining. RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups. CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.

P21-activated kinase-1 signaling is required to preserve adipose tissue homeostasis and cardiac function.

While P21-activated kinase-1 (PAK1) has been extensively studied in relation to cardiovascular health and glucose metabolism, its roles within adipose tissue and cardiometabolic diseases are less understood. In this study, we explored the effects of PAK1 deletion on energy balance, adipose tissue homeostasis, and cardiac function utilizing a whole-body PAK1 knockout (PAK1-/-) mouse model. Our findings revealed that body weight differences between PAK1-/- and WT mice emerged at 9 weeks of age, with further increases observed at 12 weeks. Furthermore, PAK1-/- mice displayed increased fat mass and decreased lean mass at 12 weeks, indicating a shift towards adiposity. In conjunction with the increased body weight, PAK1-/- mice had increased food intake and reduced energy expenditure. At a mechanistic level, PAK1 deletion boosted the expression of lipogenic markers while diminishing thermogenic markers expression in adipose tissues, contributing to reduced energy expenditure and the overall obesogenic phenotype. Moreover, our findings highlighted a significant impact on cardiac function following PAK1 deletion, including alterations in calcium kinetics and compromised systolic and lusitropy functions. In summary, our study emphasizes the significant role of PAK1 in weight regulation and cardiac function, enriching our comprehension of heart health and metabolism. These findings could potentially facilitate the identification of novel therapeutic targets in cardiometabolic diseases.

RUNX1, FUS, and ELAVL1-induced circPTPN22 promote gastric cancer cell proliferation, migration, and invasion through miR-6788-5p/PAK1 axis-mediated autophagy.

BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated. METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22's influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status. RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC. CONCLUSION: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.

Cell division cycle 42 attenuates high glucose-treated renal tubular epithelial cell apoptosis, fibrosis, and inflammation, but activates the PAK1/AKT pathway.

BACKGROUND: Cell division cycle 42 (CDC42) modulates metabolism, inflammation, and fibrosis to engage in the pathology of diabetic complications. This study intended to further investigate the influence of CDC42 on viability, apoptosis, inflammation, epithelial-mesenchymal transition, and fibrosis in high glucose (HG)-treated renal tubular epithelial cells. METHODS: HK-2 cells were exposed to HG medium (30 mM) to establish the diabetic nephropathy (DN) cellular model, then the cells were transfected with scramble overexpression control (oeNC) or CDC42 overexpression (oeCDC42) vectors. RESULTS: Both the level of CDC42 mRNA and protein were decreased in HG-treated HK-2 cells in a dose- and time-dependent manner. Then HG-treated HK-2 cells were proposed for the following experiments. It was found that CDC42 increased CCK-8 detected viability and EdU positive cells. On the contrary, CDC42 reduced cell apoptosis, which was reflected by decreased TUNEL positive rate, increased BCL2, and reduced BAX. Interestingly, CDC42 inhibited fibrosis, which was reflected by increased E-Cadherin, as well as decreased Vimentin, TGF-ß1, Collagen1, and α-SMA. Apart from these, CDC42 also attenuated proinflammatory cytokine production, including TNF-α, IL-1ß, and IL-6. Moreover, CDC42 activated the PAK1/AKT pathway, which was reflected by increased p-PAK1 and p-AKT. However, CDC42 did not affect p-ERK. CONCLUSION: CDC42 may retard DN progression via its regulation of renal tubular epithelial cell functions, which may be due to its stimulation of the PAK1/AKT pathway.

Circ_0023990 Promotes the Proliferation, Invasion, and Glycolysis of Esophageal Squamous Cell Carcinoma Cells Via Targeting miR-6884-5p/PAK1 Axis.

Circular RNAs are emerging players in human cancers, including esophageal squamous cell carcinoma (ESCC). Herein, we assessed the expression level of circ_0023990 and explored the molecular mechanisms of circ_0023990 in ESCC. circ_0023990, miR-6884-5p, and PAK1 expressions in ESCC tissues and cells were detected by quantitative real-time polymerase chain reaction and western blot. ESCC cells were transfected with different constructs to alter the expression of circ_0023990, miR-6884-5p, and PAK1. The effect of circ_0023990 on the proliferation, invasion, and glycolysis of ESCC cells was observed. The interaction between circ_0023990 and miR-6884-5p and between miR-6884-5p and PAK1 were explored. A mouse model of ESCC was established to study the in vivo effect of circ_0023990 knockdown on tumor formation.The expression levels of circ_0023990 was upregulated in ESCC tissues and cells. Inhibiting circ_0023990 suppressed the proliferation, invasion, and glycolysis of ESCC cells. circ_0023990 might target miR-6884-5p and consequently modulate the expression and activity of PAK1. Knockdown of circ_0023990 led to significantly reduced tumor volume and weight in mice with ESCC.These findings overall suggest an oncogenic role of circ_0023990 in ESCC. Future research is warranted to confirm the expression pattern and clinical significance of circ_0023990 in ESCC.

DOCK2 regulates MRGPRX2/B2-mediated mast cell degranulation and drug-induced anaphylaxis.

BACKGROUND: Drug-induced anaphylaxis is triggered by the direct stimulation of mast cells (MCs) via Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MRGPRB2). However, the precise mechanism that links MRGPRX2/B2 to MC degranulation is poorly understood. Dedicator of cytokinesis 2 (DOCK2) is a Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 regulates migration and activation of leukocytes, its role in MCs remains unknown. OBJECTIVE: We aimed to elucidate whether-and if so, how-DOCK2 is involved in MRGPRX2/B2-mediated MC degranulation and anaphylaxis. METHODS: Induction of drug-induced systemic and cutaneous anaphylaxis was compared between wild-type and DOCK2-deficient mice. In addition, genetic or pharmacologic inactivation of DOCK2 in human and murine MCs was used to reveal its role in MRGPRX2/B2-mediated signal transduction and degranulation. RESULTS: Induction of MC degranulation and anaphylaxis by compound 48/80 and ciprofloxacin was severely attenuated in the absence of DOCK2. Although calcium influx and phosphorylation of several signaling molecules were unaffected, MRGPRB2-mediated Rac activation and phosphorylation of p21-activated kinase 1 (PAK1) were impaired in DOCK2-deficient MCs. Similar results were obtained when mice or MCs were treated with small-molecule inhibitors that bind to the catalytic domain of DOCK2 and inhibit Rac activation. CONCLUSION: DOCK2 regulates MRGPRX2/B2-mediated MC degranulation through Rac activation and PAK1 phosphorylation, thereby indicating that the DOCK2-Rac-PAK1 axis could be a target for preventing drug-induced anaphylaxis.

FUBP3 regulates chronic myeloid leukaemia progression through PRC2 complex regulated PAK1-ERK signalling.

The development of resistance and heterogeneity in differential response towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) treatment has led to the exploration of factors independent of the Philadelphia chromosome. Among these are the association of deletions of genes on derivative (der) 9 chromosome with adverse outcomes in CML patients. However, the functional role of genes near the breakpoint on der (9) in CML prognosis and progression remains largely unexplored. Copy number variation and mRNA expression were evaluated for five genes located near the breakpoint on der (9). Our data showed a significant association between microdeletions of the FUBP3 gene and its reduced expression with poor prognostic markers and adverse response outcomes in CML patients. Further investigation using K562 cells showed that the decrease in FUBP3 protein was associated with an increase in proliferation and survival due to activation of the MAPK-ERK pathway. We have established a novel direct interaction of FUBP3 protein and PRC2 complex in the regulation of ERK signalling via PAK1. Our findings demonstrate the role of the FUBP3 gene located on der (9) in poor response and progression in CML with the identification of additional druggable targets such as PAK1 in improving response outcomes in CML patients.

Rac1/PAK1 signaling contributes to bone cancer pain by Regulation dendritic spine remodeling in rats.

Bone cancer pain (BCP) is severe chronic pain caused by tumor metastasis to the bones, often resulting in significant skeletal remodeling and fractures. Currently, there is no curative treatment. Therefore, insight into the underlying mechanisms could guide the development of mechanism-based therapeutic strategies for BCP. We speculated that Rac1/PAK1 signaling plays a critical role in the development of BCP. Tumor cells implantation (TCI) into the tibial cavity resulted in bone cancer-associated mechanical allodynia. Golgi staining revealed changes in the excitatory synaptic structure of WDR (Wide-dynamic range) neurons in the spinal cord, including increased postsynaptic density (PSD) length and thickness, and width of the cleft. Behavioral and western blotting test revealed that the development and persistence of pain correlated with Rac1/PAK1 signaling activation in primary sensory neurons. Intrathecal injection of NSC23766, a Rac1 inhibitor, reduced the persistence of BCP as well as reversed the remodeling of dendrites. Therefore, we concluded that activation of the Rac1/PAK1 signaling pathway in the spinal cord plays an important role in the development of BCP through remodeling of dendritic spines. Modulation of the Rac1/PAK1 pathway may be a potential strategy for BCP treatment.

Vimentin tunes cell migration on collagen by controlling ß1 integrin activation and clustering.

Vimentin is a structural protein that is required for mesenchymal cell migration and directly interacts with actin, ß1 integrin and paxillin. We examined how these interactions enable vimentin to regulate cell migration on collagen. In fibroblasts, depletion of vimentin increased talin-dependent activation of ß1 integrin by more than 2-fold. Loss of vimentin was associated with reduction of ß1 integrin clustering by 50% and inhibition of paxillin recruitment to focal adhesions by more than 60%, which was restored by vimentin expression. This reduction of paxillin was associated with 65% lower Cdc42 activation, a 60% reduction of cell extension formation and a greater than 35% decrease in cell migration on collagen. The activation of PAK1, a downstream effector of Cdc42, was required for vimentin phosphorylation and filament maturation. We propose that vimentin tunes cell migration through collagen by acting as an adaptor protein for focal adhesion proteins, thereby regulating ß1 integrin activation, resulting in well-organized, mature integrin clusters.This article has an associated First Person interview with the first author of the paper.

PLPP/CIN-mediated NF2 S10 dephosphorylation distinctly regulates kainate-induced seizure susceptibility and neuronal death through PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

BACKGROUND: Pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) selectively dephosphorylates serine (S) 10 site on neurofibromin 2 (NF2, also known as merlin (moesin-ezrin-radixin-like protein) or schwannomin). p21-activated kinase 1 (PAK1) is a serine/threonine protein kinase, which is involved in synaptic activity and plasticity in neurons. NF2 and PAK1 reciprocally regulate each other in a positive feedback manner. Thus, the aim of the present study is to investigate the effects of PLPP/CIN-mediated NF2 S10 dephosphorylation on PAK1-related signaling pathways under physiological and neuroinflammatory conditions, which are largely unknown. METHODS: After kainate (KA) injection in wild-type, PLPP/CIN-/- and PLPP/CINTg mice, seizure susceptibility, PAK1 S204 autophosphorylation, nuclear factor-κB (NF-κB) p65 S276 phosphorylation, cyclooxygenase-2 (COX-2) upregulation, prostaglandin E synthase 2 (PTGES2) induction and neuronal damage were measured. The effects of 1,1'-dithiodi-2-naphthtol (IPA-3, a selective inhibitor of PAK1) pretreatment on these responses to KA were also validated. RESULTS: PLPP/CIN overexpression increased PAK1 S204 autophosphorylation concomitant with the enhanced NF2 S10 dephosphorylation in hippocampal neurons under physiological condition. Following KA treatment, PLPP/CIN overexpression delayed the seizure on-set and accelerated PAK1 S204 phosphorylation, NF-κB p65 S276 phosphorylation, COX-2 upregulation and PTGES2 induction, which were ameliorated by PLPP/CIN deletion or IPA-3. Furthermore, IPA-3 pretreatment shortened the latency of seizure on-set without affecting seizure severity (intensity) and ameliorated CA3 neuronal death induced by KA. CONCLUSIONS: These findings indicate that PLPP/CIN may regulate seizure susceptibility (the latency of seizure on-set) and CA3 neuronal death in response to KA through NF2-PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

PAK1 c.1409 T > a (p. Leu470Gln) de novo variant affects the protein kinase domain, leading to epilepsy, macrocephaly, spastic quadriplegia, and hydrocephalus: Case report and review of the literature.

The p-21-activated kinase 1 (PAK1) protein, encoded by the PAK1 gene, is an evolutionarily conserved serine/threonine-protein kinase that regulates key cellular developmental processes. To date, seven de novo PAK1 variants have been reported to cause the Intellectual Developmental Disorder with Macrocephaly, Seizures, and Speech Delay (IDDMSSD). In addition to the namesake features, other common characteristics include structural brain anomalies, delayed development, hypotonia, and dysmorphic features. Here, we report a de novo PAK1 NM_002576.5: c.1409 T > A variant (p.Leu470Gln) identified by trio genome sequencing (GS) in a 13-year-old boy with postnatal macrocephaly, obstructive hydrocephalus, medically refractory epilepsy, spastic quadriplegia, white matter hyperintensities, profound developmental disabilities, and a horseshoe kidney. This is the first recurrently affected residue identified in the protein kinase domain. Combined assessment of the eight pathogenic PAK1 missense variants reveal that the variants cluster in either the protein kinase or autoregulatory domains. Although interpretation of the phenotypic spectrum is limited by the sample size, neuroanatomical alterations were found more often in individuals with PAK1 variants in the autoregulatory domain. In contrast, non-neurological comorbidities were found more often in individuals with PAK1 variants in the protein kinase domain. Together, these findings expand the clinical spectrum of PAK1-associated IDDMSSD and reveal potential correlations with the affected protein domains.

ATRA sensitized the response of hepatocellular carcinoma to Sorafenib by downregulation of p21-activated kinase 1.

BACKGROUND: Sorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms. METHODS: CCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues. RESULTS: We observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models. CONCLUSIONS: Our findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients. Video Abstract.

Hypermethylation of microRNA-497-3p contributes to progression of thyroid cancer through activation of PAK1/ß-catenin.

MicroRNA-497 (miR-497) has been reported to be a tumor-suppressive miRNA in thyroid cancer (TC), yet the mechanism is not clearly defined. In this study, we aim to determine the mechanism by which miR-497-3p affects the progression of TC. After characterization of low miR-497-3p expression pattern in TC and normal tissues, we assessed the correlation between miR-497-3p expression and clinicopathological features of TC patients. Its low expression shared associations with advanced tumor stage and lymph node metastasis. ChIP and methylation-specific PCR provided data showing that downregulation of miR-497-3p in TC tissues was induced by DNA methyltransferase-mediated hypermethylation. By performing dual-luciferase reporter assay, we identified that miR-497-3p targeted PAK1 while PAK1 could inhibit ß-catenin expression. Through this mechanism, miR-497-3p exerted the anti-proliferative, anti-invasive, pro-apoptotic, and anti-tumorigenic effects on TC cells on the strength of the results from gain-of-function and rescue experiments. This study suggested that hypermethylation of miR-497-3p resulted in upregulation of ß-catenin dependent on PAK1 and contributed to cancer progression in TC, which highlighted one of miR-mediated tumorigenic mechanism.