Role for p21-activated kinase PAK4 in development of the mammalian heart

The serine-threonine kinase PAK4 plays a pivotal role in cell proliferation, survival, and control of the cytoskeleton. Mice that lack Pak4 die in midgestation prior to embryonic day E11 from unidentified causes. Analysis of PAK4 protein levels demonstrated that it was highly expressed in the whole embryo and in the developing heart but became low in the hearts of adult mice. In this study we analyzed development of the heart in conventional and conditional Pak4 knockout mice and embryos. We found that in conventional Pak4 knockout mice cardiogenesis is strongly affected from early developmental stages and by E9.5, hearts of Pak4?/? embryos developed multiple profound deficits. Conditional deletion of Pak4 in the progenitors of the secondary heart field led to abnormal development of the outflow tract, in which the pulmonary artery had a smaller diameter, and the aortal wall was thinner than in wildtype mice. The conditional knockout mice also displayed the characteristic enlargement of the right ventricles and right atria. Pak4?/? embryos and cardiomyocytes in which PAK4 was depleted exhibited low levels of LIMK1, a protein that plays key roles in cytoskeletal organization. Knock down of PAK4 in cultured cardiomyocytes led to severely compromised sarcomeric structure and deficits in contraction. These results indicate that PAK4 functions, including control of actin dynamics, are necessary for normal development of the heart.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

A role for p21-activated kinase in endothelial cell migration

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and increased stress fibers and focal adhesions. The DN effect mapped to the most NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. In this report, we demonstrate an essential role for a paxillin-PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1-329) or the autoinhibitory domain (AID 83-149) induced PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. We show PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK-PIX-PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogated PAK localization. An identical result was obtained after PKLDeltaPBS2 expression. Finally, neither PAK nor PKL was capable of localizing to focal adhesions in cells overexpressing paxillinDeltaLD4, confirming a requirement for this motif in recruitment of the PAK-PIX-PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2-paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.     

Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure

During early development of the central nervous system, the neuroepithelial cells undergo dynamic changes in shape, cumulative action of which cause the neural plate to bend mediolaterally to form the neural tube. The apicobasal elongation changes the cuboidal cells into columnar ones, whereas apical constriction minimizes the cell apices, causing them to adopt wedge-like shapes. To achieve the morphological changes required for the formation of a hollow structure, these cellular changes must be controlled in time and space. To date, it is widely accepted that spatial and temporal changes of the cytoskeletal organization are fundamental to epithelial cell shape changes, and that noncetrosomal microtubules assembled along apicobasal axis and actin filaments and non-muscle myosin II at the apical side are central machineries of cell elongation and apical constriction, respectively. Hence, especially in the last decade, intracellular mechanisms regulating these cytoskeletons have been extensively investigated at the molecular level. As a result, several actin-binding proteins, Rho/ROCK pathway, and cell-cell adhesion molecules have been proven to be the central regulators of apical constriction, while the regulatory mechanisms of cell elongation remain obscure. In this review, we first describe the distribution and role of cytoskeleton in cell shape changes during neural tube closure, and then summarize the current knowledge about the intracellular proteins that directly modulate the cytoskeletal organization and thus the neural tube closure.     

A key role for Pak4 in proliferation and differentiation of neural progenitor cells

The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4 weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain.     

The effects of phorbol ester on mouse blastomeres: a role for protein kinase C in compaction?

The effects of phorbol myristate acetate (PMA) and other activators of protein kinase C on the cytoskeletal organization of mouse oocytes and early embryos have been examined. The effects observed depended on the developmental stage on exposure to PMA. PMA had little effect on the cytoskeletal or microvillous organization of unfertilized oocytes. Interphase cells from embryos prior to compaction showed limited disruption and loss of microvilli when exposed to PMA and foci of polymerized actin remained visible in the cytocortex of embryos up to the early 8-cell stage. When compacted late 8-cell embryos were exposed to PMA, most microvilli were lost and little polymerized actin remained in the cytocortex. PMA also caused loss of microtubules from compact 8-cell embryos under some experimental conditions. Intercellular flattening was both prevented and reversed. The relevance of these observations to the rearrangement of cell-cell contacts and cytoskeletal organization seen during compaction at the 8-cell stage is discussed and a possible role for protein kinase C in the generation of cell polarity proposed.     

p21活化激酶1在口腔癌细胞侵袭和迁移中的作用

p21活化激酶1(PAK1)与多种癌症类型的进展有关,然而,其在口腔癌中的作用仍不明确。本研究以人舌鳞状细胞癌(oral squamous cell carcinoma,OSCC)HSC4细胞系为研究材料,考察了PAK1在细胞系中的定位及血清生长因子对PAK1定位的影响。通过转染PAK1 siRNA来敲低OSCC细胞中的PAK1,并分析PAK1敲低对细胞侵袭和迁移性的影响。研究发现PAK1在人舌鳞状细胞癌细胞系HSC4中主要定位于细胞核,而血清生长因子可调节PAK1在不同亚细胞区域中的易位或积累,并且还可能影响OSCC细胞的细胞骨架结构。此外,敲低PAK1可显著抑制OSCC细胞的迁移和侵袭能力,表明PAK1在OSCC发生发展过程中起着至关重要的作用。     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.     

Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation

Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.     

An adenosine triphosphatase of the sucrose nonfermenting 2 family, androgen receptor-interacting protein 4, is essential for mouse embryonic development and cell proliferation

An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4-/- embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4-/- embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4-/- embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4-/- and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Ultrastructural analysis of malformations of the embryonic neural axis induced by in vitro hyperglycemic conditions

Neural tube defects are the most common malformations associated with diabetic pregnancies. Although the teratogenic effects of excess glucose have been investigated in in vivo and in vivo studies, a cellular basis for neural tube defects has not been elucidated. We used rat embryo culture to study the organogenesis period of development, with excess d-glucose added to the serum medium to induce neural tube anomalies. Light and electron microscopic examination of control 12-day-old embryos grown 48 hours in culture revealed blastlike cells with few organelles or cellular processes. Twelve-day-old embryos cultured in excess d-glucose had advanced cellular maturation with differentiation, including the presence of free polysomes and copious cell processes, regardless of whether they had an open neural tube. Cytoarchitectural changes such as decreased numbers of mitotic figures with mitotic cells in the mantle layer were focally distributed throughout the neural epithelium but with predominance at the site of failed closure. In vivo studies failed to demonstrate neural processes in day 12 normal embryos. Fourteen-day-old embryos grown in utero also had foci of cell processes in the neural tube but to a much lesser degree than that observed in the in vitro day 12 glucose-exposed embryos. The cellular aberrations in the excess d-glucose-treated embryos are characteristic of a premature maturational change. Since they are present in excess d-glucose-exposed embryos with or without failure of neural tube closure, these maturational and cytoarchitectural changes may contribute to the cellular basis for neural tube defects.     

P21-activated kinase 4 overexpression in metastatic gastric cancer patients

INTRODUCTION: P21-activated kinase 4 (PAK), a subfamily of serine/threonine kinases originally known as a regulator of cytoskeletal dynamics and cell motility, has recently been revealed to play a key role in oncogenic signaling pathways. We studied the frequency and clinical features of PAK4-overexpressed metastatic gastric cancer. PATIENTS AND METHODS: PAK4 overexpression was screened by Western blot in 18 human gastric cancer cell lines. Immunohistochemical staining of PAK4 protein was performed in tumor specimens of 49 metastatic gastric cancer patients who received palliative capecitabine/cisplatin as first-line treatment. RESULTS: PAK4 protein overexpression was detected strongly in five gastric cell lines (AGS, MGK-28, MKN-74, SNU-216, SNU-601) and weakly in four cell lines (KATOIII, MKN-1, SNU-620, and SNU-719). PAK4 knockdown by small interfering RNA induced apoptosis in PAK4-overexpressed AGS gastric cancer cells. Immunohistochemical staining revealed PAK4 overexpressions in 4 (8.1%) of 49 metastatic gastric cancer specimens. None of the four patients with PAK4(+) responded to capecitabine/cisplatin chemotherapy, and PAK4(+) gastric cancer patients had a trend of poorer survival compared with PAK(-)(P = .876). CONCLUSIONS: We demonstrated PAK4 overexpression in a subset of gastric cancer patients, implicating a role in gastric cancer tumorigenesis. Its prognostic significance and efficacy as a drug target should be further studied.     

DLC-1, a Rho GTPase-activating protein with tumor suppressor function, is essential for embryonic development

DLC-1 (deleted in liver cancer 1) is a Rho GTPase-activating protein that is able to inhibit cell growth and suppress tumorigenesis. We have used homologous recombination to inactivate the mouse DLC-1 gene (Arhgap7). Mice heterozygous for the targeted allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Histological analysis revealed that DLC-1-/- embryos had defects in the neural tube, brain, heart, and placenta. Cultured fibroblasts from DLC-1-deficient embryos displayed alterations in the organization of actin filaments and focal adhesions.