Deletion Of Pak1 Research Articles

Deletion of Pak1 in CD11c-Positive Cells Confers Resistance to Mouse Skin Carcinogenesis

Deletion of Pak1 in CD11c-Positive Cells Confers Resistance to Mouse Skin Carcinogenesis

Inhibition of PAK1 generates an ameliorative effect on MPLW515L mouse model of myeloproliferative neoplasms by regulating the differentiation and survival of megakaryocytes

Most thrombopoietin receptor (MPL) mutations result in abnormal megakaryocyte expansion in the spleen or bone marrow (BM), leading to progressive fibrosis. It has been reported that p21 (RAC1)-activated kinase 1 (PAK1) participates in the proliferation and differentiation of megakaryoblast, PAK1 phosphorylation was found to be increased in patients with myeloproliferative neoplasm (MPNs) and murine MPN cells with the MPLW515L mutant gene in the present study; however, the function of the over activated PAK1 in MPN cells remains unclear. We found that inhibition of PAK1 caused significant changes in the biological behaviors of MPLW515L mutant cells in vitro, including arrested growth or reduced clonality as well as increased polyploid DNA and cells apoptosis due to upregulated cleaved caspase 3. In vivo, PAK1 inhibitor treatment caused a slow elevation of leukocytosis and hematocrit (HCT) and reduction in hepatosplenomegaly in 6133/MPLW515L transplanted mice, along with reduced tumor cell infiltration and prolonged survival. Further deletion of PAK1 sustained a relatively normal HCT and platelet count at the beginning of disease but did not completely alleviate the splenomegaly of MPLW515L mutant mice. Notably, PAK1 knockout attenuated the destruction of splenic structure, and reduced the megakaryocyte burden within the BM. These results suggest that the inhibition of PAK1 may be a considerable method treatingMPLW515L mutant MPN by intervening megakaryocytes.

PAK1 inhibition reduces tumor size and extends the lifespan of mice in a genetically engineered mouse model of Neurofibromatosis Type 2 (NF2).

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.

P21-activated kinase 1 restricts tonic endocannabinoid signaling in the hippocampus.

PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies.

A novel PAK1/Notch-1 axis contributes to intestinal inflammation and cancer

Background: PAK1 belongs to a family of serine-threonine kinase and contributes to various oncogenic pathways like MAPK and Wnt-β catenin. Our lab recently linked activation of p21-activated kinase (PAK1) to IBD as well as colitis-associated cancer (Khare V, Inflamm Bowel Dis 2015). Here we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL-10 KO mice. Methods: IL-10 KO mice were crossed with Pak1 KO mice to generate double-knockout mice (DKO). Inflammation was triggered with piroxicam (200ppm mixed to chow for 14 d). After 12 wks mice were euthanized, intestines were prepared and length was measured. Paraffin embedded Swiss rolls were stained with HE MAPK pathway activation, as shown by increased pERK1/2 staining and an increase in γH2AX positive nuclei, indicating more DNA double-strand breaks. A reduced differentiation of goblet cells and Paneth cells was marked by PAS- and Lysozyme staining, together with an expansion of the stem cell compartment (shown by increased activated Notch1 and LGR5 staining) in the colonic crypts of DKOs. Interestingly NFκB was found to be reduced in DKO mice. Conclusion: Our data suggest a novel interaction of PAK-1 with the Notch pathway in regulating crypt cell stemness, proliferation and differentiation. Lack of mature goblet cells and mucus can exacerbate colitis in IL-10 KO mice. Hyperproliferation may promote dysplastic lesions independent of NFκB.

PAK1 promotes intestinal tumor initiation.

p21-activated kinase 1 (PAK1) is a serine/threonine kinase that is overexpressed in colorectal cancer. PAK1 is a target of mesalamine [5-aminosylicylic acid (5-ASA)], a common drug for the treatment of ulcerative colitis with prospective chemopreventive properties. Here, we investigated whether PAK1 deletion impedes tumorigenesis in murine intestinal cancer models. Ten-week-old APC(min) or APC(min)/PAK1(-/-) mice were monitored for 8 weeks, euthanized, and assessed for tumor number and size. Six- to 8-week-old PAK1(-/-) and wild-type (WT) mice received one 10 mg/kg intraperitoneal injection of azoxymethane (AOM) and four cycles of 1.7% dextran sodium sulfate (DSS) for 4 days followed by 14 days of regular water. Mice also received 5-ASA via diet. Tumor incidence and size was assessed via colonoscopy and pathology. Molecular targets of PAK1 and 5-ASA were evaluated via immunohistochemistry (IHC) in both models. PAK1 deletion reduced tumor multiplicity and tumor burden but did not alter average tumor size in APC(min) mice. IHC revealed that PAK1 deletion reduced p-AKT, β-catenin, and c-Myc expression in APC(min) adenomas. Colonoscopy and pathologic analysis revealed that PAK1 deletion reduced tumor multiplicity without affecting tumor size in AOM/DSS-treated mice. 5-ASA treatment and PAK1 deletion impeded tumor multiplicity and dysplastic lesions in AOM/DSS mice. IHC further revealed that 5-ASA blocked β-catenin signaling via inhibition of PAK1/p-AKT. These data indicate that PAK1 contributes to initiation of intestinal carcinogenesis.

Polycomb-mediated loss of microRNA let-7c determines inflammatory macrophage polarization via PAK1-dependent NF-κB pathway.

Serine/threonine kinase family members p21-activated kinases (PAKs) are important regulators of cytoskeletal remodeling and cell motility in mononuclear phagocytic system, but their role in macrophage differentiation and polarization remains obscure. We have shown here that inflammatory stimuli induced PAK1 overexpression in human and murine macrophages. Elevated expression of PAK1 contributed to macrophage M1 polarization and lipopolysaccharide (LPS)-induced endotoxin shock. We further observed that epigenetic loss of microRNA let-7c due to enhancer of zeste homolog 2 (EZH2) upregulation determined PAK1 elevation and inflammatory phenotype in M1 macrophages. EZH2/let-7c/PAK1 axis promotes macrophage M1 polarization via NIK-IKK-NF-κB signaling. Moreover, pharmacological and genetic ablation with EZH2/let-7c/PAK1 axis blunted inflammatory phenotype in M1 macrophages. Critically, either myeloid-restricted PAK1 deletion (PAK1(Lyz2cre)) or pharmacological and genetic ablation with EZH2/let-7c/PAK1 signal resulted in resistance to LPS-induced endotoxin shock via blunting macrophage M1 polarization. PAK1, therefore, is an essential controller of inflammatory macrophage polarization, regulating immune responses against pathogenic stimuli.

P21-Activated Protein Kinase 1 (Pak1) Mediates the Cross Talk between Insulin and β-Catenin on Proglucagon Gene Expression and Its Ablation Affects Glucose Homeostasis in Male C57BL/6 Mice

In gut endocrine L cells, the Wnt signaling pathway effector β-catenin (β-cat)/transcription factor 7-like 2 mediates the stimulatory effect of insulin on proglucagon (gcg) expression and glucagon-like peptide-1 (GLP-1) production. In several other cell lineages, insulin is able to stimulate p21-activated protein kinase 1 (Pak1). Here we determined the role of Pak1 in gcg expression and the effect of Pak1 deletion on glucose homeostasis. Insulin stimulated Pak1 activation through increasing its Thr423 phosphorylation in gut gcg-expressing cell lines, associated with increased gcg mRNA levels. This stimulation was attenuated by the Pak inhibitor 2,2'-dihydroxy-1,1'-dinaphthyldisulfide (IPA3) or dominant-negative Pak1. Both insulin and cAMP-promoting agents activated β-cat Ser675 phosphorylation, which was attenuated by IPA3 or protein kinase A inhibition, respectively. Gut gcg levels were reduced in male Pak1(-/-) mice, associated with impaired glucose tolerance after an ip or oral glucose challenge. These mice had lower circulating active GLP-1 levels after a glucose challenge as well as reduced distal ileum GLP-1 content after insulin treatment. Finally, the Pak1(-/-) mice exhibited reduced brainstem gcg level and abolished β-cat Ser675 phosphorylation in brain neurons after insulin treatment. We suggest that Pak1 mediates the cross talk between insulin and Wnt signaling pathways on gut and brain gcg expression, and its ablation impairs glucose homeostasis.

Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1 −/−) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1 −/− and wild type mice. Pak1 −/− mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1 −/− mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.

Pak1 as a Novel Therapeutic Target for Antihypertrophic Treatment in the Heart

Stress-induced hypertrophic remodeling is a critical pathogenetic process leading to heart failure. Although many signal transduction cascades are demonstrated as important regulators to facilitate the induction of cardiac hypertrophy, the signaling pathways for suppressing hypertrophic remodeling remain largely unexplored. In this study, we identified p21-activated kinase 1 (Pak1) as a novel signaling regulator that antagonizes cardiac hypertrophy. Hypertrophic stress applied to primary neonatal rat cardiomyocytes (NRCMs) or murine hearts caused the activation of Pak1. Analysis of NRCMs expressing constitutively active Pak1 or in which Pak1 was silenced disclosed that Pak1 played an antihypertrophic role. To investigate the in vivo role of Pak1 in the heart, we generated mice with a cardiomyocyte-specific deletion of Pak1 (Pak1(cko)). When subjected to 2 weeks of pressure overload, Pak1(cko) mice developed greater cardiac hypertrophy with attendant blunting of JNK activation compared with controls, and these knockout mice underwent the transition into heart failure when prolonged stress was applied. Chronic angiotensin II infusion also caused increased cardiac hypertrophy in Pak1(cko) mice. Moreover, we discovered that the Pak1 activator FTY720, a sphingosine-like analog, was able to prevent pressure overload-induced hypertrophy in wild-type mice without compromising their cardiac functions. Meanwhile, FTY720 failed to exert such an effect on Pak1(cko) mice, suggesting that the antihypertrophic effect of FTY720 likely acts through Pak1 activation. These results, for the first time, establish Pak1 as a novel antihypertrophic regulator and suggest that it may be a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.

Activation of Pak1/Akt/eNOS signaling following sphingosine-1-phosphate release as part of a mechanism protecting cardiomyocytes against ischemic cell injury

We investigated whether plasma long-chain sphingoid base (LCSB) concentrations are altered by transient cardiac ischemia during percutaneous coronary intervention (PCI) in humans and examined the signaling through the sphingosine-1-phosphate (S1P) cascade as a mechanism underlying the S1P cardioprotective effect in cardiac myocytes. Venous samples were collected from either the coronary sinus (n = 7) or femoral vein (n = 24) of 31 patients at 1 and 5 min and 12 h, following induction of transient myocardial ischemia during elective PCI. Coronary sinus levels of LCSB were increased by 1,072% at 1 min and 941% at 5 min (n = 7), while peripheral blood levels of LCSB were increased by 579% at 1 min, 617% at 5 min, and 436% at 12 h (n = 24). In cultured cardiac myocytes, S1P, sphingosine (SPH), and FTY720, a sphingolipid drug candidate, showed protective effects against CoCl induced hypoxia/ischemic cell injury by reducing lactate dehydrogenase activity. Twenty-five nanomolars of FTY720 significantly increased phospho-Pak1 and phospho-Akt levels by 56 and 65.6% in cells treated with this drug for 15 min. Further experiments demonstrated that FTY720 triggered nitric oxide release from cardiac myocytes is through pertussis toxin-sensitive phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase signaling. In ex vivo hearts, ischemic preconditioning was cardioprotective in wild-type control mice (Pak1f/f), but this protection appeared to be ineffective in cardiomyocyte-specific Pak1 knockout (Pak1cko) hearts. The present study provides the first direct evidence of the behavior of plasma sphingolipids following transient cardiac ischemia with dramatic and early increases in LCSB in humans. We also demonstrated that S1P, SPH, and FTY720 have protective effects against hypoxic/ischemic cell injury, likely a Pak1/Akt1 signaling cascade and nitric oxide release. Further study on a mouse model of cardiac specific deletion of Pak1 demonstrates a crucial role of Pak1 in cardiac protection against ischemia/reperfusion injury.

P21-Activated Kinase 1 Regulates Hyperproliferation of Nf1 Haploinsufficient Mast Cells In Vitro and In Vivo Via Activation of the MAPK Pathway.

p21-activated kinases (Paks) are downstream mediators of Rho GTPase proteins and have been implicated in yeast and immortalized cells as positive regulators of MAPK pathway members in modulating cell growth and cytoskeletal functions. However, their role in primary mammalian cells has not been described.NF1 encodes neurofibromin, which negatively regulates p21Ras activity by stimulating its intrinsic GTPase activity, and accelerating hydrolysis of Ras from the GTP to the GDP confirmation. Disruption of the NF1 locus results in neurofibromatosis type I (NF1), an inherited disorder characterized by the development of neurofibromas that contain large numbers of degranulating mast cells that have been implicated in tumor progression. Utilizing a genetic intercross of Pak 1−/− mice with mice haploinsufficient at the Nf1 locus, we studied the role of Pak1 in the context of normal and hyperactivated Ras-MAPK signaling in primary inflammatory mast cells. Pak1 was found to directly contribute to Ras-dependent signaling by modulating both Raf-1, Mek-1 and ERK1/2 activation. Loss of Pak1 fully corrects the hyperphosphorylation of ERK1/2 found in Nf1+/− mast cells to that of wild type controls. Deletion of Pak1 in Nf1+/− mast cells is associated with a correction of Kit ligand mediated proliferation to wild type levels in vitro. Further, after subcutaneous administration of Kit ligand via micro osmotic pumps, which is an established model that stimulates local proliferation of mast cells in vivo (Ingram, JEM 2001), we confirmed that genetic disruption of Pak1 corrects the proliferation of Nf1+/− mast cells in vivo to that of wild type controls. These data provide direct genetic evidence that Pak1 modulates the Ras-Raf-Mek-Erk pathway and identifies a specific molecular target within the inflammatory tumor microenvironment for the treatment or prevention of neurofibromas.

Characterization of Pak2p, a Pleckstrin Homology Domain-containing, p21-activated Protein Kinase from Fission Yeast

p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.