Conditional Knockout of N-WASP Enhanced the Formation of Keratinizing Squamous Cell Carcinoma Induced by KRasG12D.

Squamous cell carcinoma (SCC) is one of the most common forms of skin cancer in humans, and Neural Wiskott-Aldrich Syndrome Protein (N-WASP) plays a crucial role in epidermal homeostasis. To elucidate the role of N-WASP in skin cancer, we generated mice which expressed constitutively active KRas (KRasG12D) in keratinocytes with either homozygous (N-WASPKOG12D) or heterozygous (N-WASPHetG12D) N-WASP knockout upon Tamoxifen (TAM) injection. Both the N-WASPKOG12D and N-WASPHetG12D mice had similar body weights and no congenital malformations prior to the injection of TAM. Within 2 weeks of the injections, the N-WASPKOG12D mice exhibited significant reductions in weight coupled with visible tumors at numerous sites, unlike the N-WASPHetG12D mice, which had no visible tumors. We found that both sets of mice had oily, sticky skin and wet eyes 3 weeks after their exposure to TAM, indicating the overproduction of sebum/meibum. At 37 days post TAM injection, several notable observations were made. Tumors collected from the N-WASPKOG12D mice had small- to large-sized keratin pearls that were not observed in the N-WASPHetG12D mice. A Western blot and immunostaining analysis both highlighted significantly higher levels of expression of SCC markers, such as the cytokeratins 8, 17, 18, and 19 and TP63, in the tumors of the N-WASPKOG12D mice compared to those of the latter group. Furthermore, we noted increases in the expression levels of EGFR, P-ERK, GLUT1, P-mTOR, and P-4EBP in the N-WASPKOG12D mice, suggesting that the deletion of N-WASP in the keratinocytes enhanced KRas signaling and glucose uptake, resulting in aggressive tumor formation. Interestingly, a thickening of the epidermal layer within the esophagus and tongue was only observed in the N-WASPKOG12D mice. Immunostaining for PCNA emphasized a significantly higher number of PCNA-positive cells in the skin of the N-WASPKOG12D mice compared to their counterparts, implying that epidermal thickening and enhanced tumorigenesis are due to an increased proliferation of keratinocytes. Through our results, we have established that N-WASP plays a tumor-suppressive role in skin cancer.

Olive-Derived Antioxidant Dietary Fiber Modulates Gut Microbiota Composition and Attenuates Atopic Dermatitis Like Inflammation in Mice.

SCOPE: Epidemiological data suggest that altered gut microbiota contributes to the development of atopic dermatitis (AD). The effect of an olive-derived antioxidant dietary fiber (OADF) in relieving AD symptoms in a murine model of 2,4-dinitrofluorobenzene (DNFB)-induced AD is examined and the effect of OADF in modulating host gut microbiota is explored. METHODS AND RESULTS: Mice are fed with either standard diet or standard diet + OADF for 3 weeks prior to induction of AD and maintained on the same diet throughout the DNFB application period. Dietary OADF causes significant improvement of AD-like symptoms with reduced serum levels of immunoglobulin (Ig)E, interleukin (IL)-1ß, IL-6, C-X-C motif ligand (CXCL)1, and increased serum levels of IL-10. OADF supplementation restore gut microbiota composition that are altered in AD mice. Specifically, OADF increases the proportion of intestinal bacteria (Ruminococcaceae UCG014, GCA900066575, UBA1819) associated with enhanced butyrate production, along with inhibiting Clostridiales vadin BB60 which are more prevalent in AD mice. CONCLUSION: OADF modulates gut microbiota composition, improves cytokine profile and butyrate production influencing AD-associated immune response. Results highlight the importance of the gut-skin axis for the AD dietary therapeutic agents.

N-WASP Attenuates Cell Proliferation and Migration through ERK2-Dependent Enhanced Expression of TXNIP.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP) regulates actin cytoskeleton remodeling. It has been known that reduced N-WASP expression in breast and colorectal cancers is associated with poor prognosis. Here, we found reduced N-WASP expression in squamous cell carcinoma (SCC) patient samples. The SCC cell line HSC-5 with reduced N-WASP expression was used to generate HSC-5CN (control) and HSC-5NW (N-WASP overexpression) cells. HSC-5NW cells had reduced cell proliferation and migration compared to HSC-5CN cells. HSC-5NW cells had increased phospho-ERK2 (extracellular signal-regulated kinase 2), phosphorylated Forkhead box protein class O1 (FOXO1) and reduced nuclear FOXO1 staining compared to HSC-5CN cells. Proteasome inhibition stabilized total FOXO1, however, not nuclear staining, suggesting that FOXO1 could be degraded in the cytoplasm. Inhibition of ERK2 enhanced nuclear FOXO1 levels and restored cell proliferation and migration of HSC-5NW to those of HSC-5CN cells, suggesting that ERK2 regulates FOXO1 activity. The expression of thioredoxin-interacting protein (TXNIP), a FOXO1 target that inhibits thioredoxin and glucose uptake, was higher in HSC-5NW cells than in HSC-5CN cells. Knockdown of TXNIP in HSC-5NW cells restored cell proliferation and migration to those of HSC-5CN cells. Thus, we propose that N-WASP regulates cell proliferation and migration via an N-WASP-ERK2-FOXO1-TXNIP pathway.

Catalase-Integrated Hyaluronic Acid as Nanocarriers for Enhanced Photodynamic Therapy in Solid Tumor.

Photodynamic therapy (PDT) as a treatment method has many advantages such as minimal invasiveness, repeatable dosage, and low systemic toxicity. Issues with conventional PDT agents include the limited availability of endogenous oxygen and difficulty in accumulation at the tumor site, which has hindered the successful treatment of tumors. Herein, we developed catalase-encapsulated hyaluronic-acid-based nanoparticles loaded with adamantane-modified photosensitizer for enhanced PDT of solid tumors. Chlorin e6 (Ce6) as the photosensitizer was modified with adamantane to yield adamantane-modified Ce6 (aCe6). The obtained nanosystem (HA-CAT@aCe6) could target overly expressed CD44 receptors on cancer cells, supplying oxygen by converting endogenous hydrogen peroxide (H2O2) to oxygen, and improving PDT efficacy upon light irradiation. HA-CAT@aCe6 nanoparticles showed high colloidal stability and monodispersity in aqueous solution. The uptake and targeting property of HA-CAT@aCe6 were demonstrated by confocal microscopy and flow cytometry in the MDA-MB-231 cell line possessing overly expressed CD44 receptors. The encapsulated catalase was able to decompose the endogenous H2O2 to generate O2 in situ for relieving hypoxia in cells incubated under hypoxic conditions. Cell viability assays indicated that HA-CAT@aCe6 possessed minimal cytotoxicity in the dark, while presenting high cellular toxicity under 660 nm light irradiation at normoxic conditions. As a result of the catalase capability in relieving hypoxia, HA-CAT@aCe6 also exhibited high cellular cytotoxicity under hypoxic condition. In vivo experiments revealed selective tumor accumulation of HA-CAT@aCe6 in MDA-MB-231 tumor bearing nude mice. Significant tumor regression was observed after intravenous injection of HA-CAT@aCe6 under light irradiation in comparison to the control system without loading catalase. Thus, HA-CAT@aCe6 demonstrated a great potential in overcoming hypoxia for targeted PDT.

Overexpression of CDC42SE1 in A431 Cells Reduced Cell Proliferation by Inhibiting the Akt Pathway.

Cell division cycle 42 (CDC42), a small Rho GTPase, plays a critical role in many cellular processes, including cell proliferation and survival. CDC42 interacts with the CRIB (Cdc42- and Rac-interactive binding) domain of CDC42SE1, a small effector protein of 9 kDa. We found that the expression of CDC42SE1 was reduced in human skin cancer samples relative to matched perilesional control. Exogenous expression of CDC42SE1 but not CDC42SE1H38A (mutation within CRIB domain) in A431 cells (A431SE1, A431SE1-H38A) reduced cell proliferation. Antibody microarray analysis of A431Ctrl and A431SE1 lysate suggested that reduced A431SE1 cells proliferation was due to inhibition of Akt pathway, which was confirmed by the reduced P-Akt and P-mTOR levels in A431SE1 cells compared to A431Ctrl cells. This suggests that CDC42SE1 modulates the CDC42-mediated Akt pathway by competing with other effector proteins to bind CDC42. A431SE1 cells formed smaller colonies in soft agar compared to A431Ctrl and A431SE1-H38A cells. These findings correlate with nude mice xenograft assays, where A431SE1 cells formed tumors with significantly-reduced volume compared to the tumors formed by A431Ctrl cells. Our results suggest that CDC42SE1 is downregulated in skin cancer to promote tumorigenesis, and thus CDC42SE1 might be an important marker of skin cancer progression.

Overexpression of GRB2 Enhances Epithelial to Mesenchymal Transition of A549 Cells by Upregulating SNAIL Expression.

GRB2 is an adaptor protein which interacts with phosphorylated TGF-ß receptor and is critical for mammary tumour growth. We found that TGF-ß1-induced EMT increased GRB2 expression in A549 cells (non-small cell lung cancer). Overexpression of GRB2 (A549GRB2) enhanced cell invasion while knocking down GRB2 (A549GRB2KD) reduced cell migration and invasion, probably due to increased vinculin and reduced Paxillin patches in A549GRB2KD cell. TGF-ß1-induced EMT was more pronounced in A549GRB2 cells and attenuated in A549GRB2KD cells. This could be due to the reduced expression of E-cadherin in A549GRB2 and increased expression of E-cadherin in A549GRB2KD cells, even before TGF-ß1 stimulation. Expression of SNAIL was elevated in A549GRB2 cells and was further enhanced by TGF-ß1 stimulation, suggesting that GRB2 down-regulates E-cadherin by enhancing the expression of SNAIL. The N-SH3 domain of GRB2 was critical for suppressing E-cadherin expression, while the C-SH3 domain of GRB2 mediating interaction with proteins such as N-WASP was critical for promoting invasion, and the SH2 domain was critical for suppressing E-cadherin expression and invasion. Thus, our data suggests that GRB2 enhances EMT by suppressing E-cadherin expression and promoting invasion probably through N-WASP to promote metastasis.

Expression of N-WASP is regulated by HiF1α through the hypoxia response element in the N-WASP promoter.

Cancer cell migration and invasion involves temporal and spatial regulation of actin cytoskeleton reorganization, which is regulated by the WASP family of proteins such as N-WASP (Neural- Wiskott Aldrich Syndrome Protein). We have previously shown that expression of N-WASP was increased under hypoxic conditions. In order to characterize the regulation of N-WASP expression, we constructed an N-WASP promoter driven GFP reporter construct, N-WASPpro-GFP. Transfection of N-WASPpro-GFP construct and plasmid expressing HiF1α (Hypoxia Inducible factor 1α) enhanced the expression of GFP suggesting that increased expression of N-WASP under hypoxic conditions is mediated by HiF1α. Sequence analysis of the N-WASP promoter revealed the presence of two hypoxia response elements (HREs) characterized by the consensus sequence 5'-GCGTG-3' at -132 bp(HRE1) and at -662 bp(HRE2) relative to transcription start site (TSS). Site-directed mutagenesis of HRE1(-132) but not HRE2(-662) abolished the HiF1α induced activation of N-WASP promoter. Similarly ChIP assay demonstrated that HiF1α bound to HRE1(-132) but not HRE2(-662) under hypoxic condition. MDA-MB-231 cells but not MDA-MB-231KD cells treated with hypoxia mimicking agent, DMOG showed enhanced gelatin degradation. Similarly MDA-MB-231KD(N-WASPpro-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of WT N-WASPpro but not MDA-MB-231KD(N-WASPproHRE1-N-WASPR) cells expressing N-WASPR under the transcriptional regulation of N-WASPproHRE1 showed enhanced gelatin degradation when treated with DMOG. Thus indicating the importance of N-WASP in hypoxia induced invadopodia formation. Thus, our data demonstrates that hypoxia-induced activation of N-WASP expression is mediated by interaction of HiF1α with the HRE1(-132) and explains the role of N-WASP in hypoxia induced invadopodia formation.

Conditional knock out of N-WASP in keratinocytes causes skin barrier defects and atopic dermatitis-like inflammation.

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously and regulates actin cytoskeleton remodeling. In order to characterize the role of N-WASP in epidermal homeostasis and cutaneous biology, we generated conditional N-WASP knockout mouse using CK14-cre (cytokeratin 14) to ablate expression of N-WASP in keratinocytes. N-WASPK14KO (N-WASP fl/fl ; CK14-Cre) mice were born following Mendelian genetics suggesting that N-WASP expression in keratinocytes is not essential during embryogenesis. N-WASPK14KO mice exhibited stunted growth, alopecia, dry and wrinkled skin. The dry skin in N-WASPK14KO mice is probably due to increased transepidermal water loss (TEWL) caused by barrier function defects as revealed by dye penetration assay. N-WASPK14KO mice developed spontaneous inflammation in the neck and face 10 weeks after birth. Histological staining revealed thickening of the epidermis, abnormal cornified layer and extensive infiltration of immune cells (mast cells, eosinophils and T-lymphocytes) in N-WASPK14KO mice skin compared to control mice. N-WASPK14KO mice had higher serum levels of IL-1α, TNF-α, IL-6 and IL-17 compared to control mice. Thus our results suggest that conditional N-WASP knockout in keratinocytes leads to compromised skin barrier, higher infiltration of immune cells and hyperproliferation of keratinocytes due to increased production of cytokines highlighting the importance of N-WASP in maintaining the skin homeostasis.

Myogenic differentiation depends on the interplay of Grb2 and N-WASP.

Myogenesis requires a well-coordinated withdrawal from cell cycle, morphological changes and cell fusion mediated by actin cytoskeleton. Grb2 is an adaptor protein whose central SH2 domain binds to phosphorylated tyrosine residues of activated receptors and activates intracellular signaling pathway, while its N-terminal and C-terminal SH3 domains bind to proline rich proteins such as N-WASP (Neural-Wiskott Aldrich Syndrome Protein). We found that the expression of Grb2 was increased at the beginning of differentiation and remained constant during differentiation in C2C12 myoblasts. Knocking down endogenous Grb2 expression caused a significant increase in the fusion index and expression of MyHC, a terminal differentiation marker when compared with the control. Over expression of Grb2 in C2C12 (C2C12Grb2-Myc) reduced myotube formation and expression of MyHC. Similarly over expression of Grb2P49L-Myc (N-terminal SH3 domain mutant) or Grb2R86K-Myc (SH2 domain mutant) inhibited myogenic differentiation of C2C12 cells. However, the expression of Grb2P206L-Myc (C-terminal SH3 domain mutant) did not inhibit myotube formation and expression of MyHC. This suggests that the C-terminal SH3 domain of Grb2 is critical for the inhibition of myogenic differentiation. The C2C12Grb2-Myc cells have reduced phalloidin staining at late stages of differentiation. Expression of N-WASP in C2C12Grb2-Myc cells rescued the myogenic defect and increased phalloidin staining (increased F-actin) in these cells. Thus our results suggest that Grb2 is a negative regulator of myogenesis and reduces myogenic differentiation by inhibiting actin polymerization/remodeling through its C-terminal SH3 domain.

WIP promotes in-vitro invasion ability, anchorage independent growth and EMT progression of A549 lung adenocarcinoma cells by regulating RhoA levels.

Cancer cell migration and invasion involves actin cytoskeleton reorganization, which is regulated by the WASP (Wiskott Aldrich Syndrome Protein) family of proteins such as WASP, N-WASP (Neural-WASP) and WASP interacting protein (WIP). In this study, we found that the expression of WIP was significantly upregulated in metastatic A5-RT3 cells compared to its parental non-tumorigenic HaCaT cells. Using A549 human lung adenocarcinoma cell line as the model system, we found that WIP regulates cell invasion, proliferation and anchorage-independent growth. Expression of WIP was enhanced during TGF-ß1 induced epithelial-mesenchymal transition (EMT) and overexpression of WIP accelerated EMT while knocking down WIP attenuated EMT associated morphological changes. Knocking down WIP expression in A549 cells significantly reduced RhoA levels and WIP was found to interact with RhoA suggesting that WIP might be executing its function by regulating RhoA. Acquisition of invasive, proliferative properties and anoikis resistance is the central step in metastasis indicating a novel function of WIP in cancer progression.

Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure.

Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFß signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice.

Molecular difference between WASP and N-WASP critical for chemotaxis of T-cells towards SDF-1α.

Wiskott-Aldrich Syndrome protein (WASP) integrates cell signaling pathways to the actin cytoskeleton, which play a critical role in T-cell activation and migration. Hematopoietic cells express both WASP and neural-WASP (N-WASP) which share similar domain structure, yet WASP deficiency causes Wiskott-Aldrich syndrome, suggesting that N-WASP present in the cells is not able to carry out all the functions of WASP. We have identified a unique internal thirty amino acid region (I30) in WASP, which regulates its function in chemotaxis of Jurkat T-cells. Deletion of the I30 region altered the WASP's closed conformation and impaired its ability to rescue the chemotactic defect of WASP-deficient (Jurkat(WKD)) T-cells. Expression of N-WASP in Jurkat(WKD) T-cells using WASP promoter restored the migration velocity without correcting the chemotactic defect. However, insertion of I30 region in N-WASP (N-WASP-I30) enabled N-WASP to rescue the chemotactic defect of Jurkat(WKD) T-cells. N-WASP-I30-EGFP displayed a punctate localization in contrast to the predominant nuclear localization of N-WASP-EGFP. Thus, our study has demonstrated that the I30 region of WASP is critical for localization and chemotaxis. This suggests that N-WASP's failure to compensate for WASP in rescuing chemotaxis could be due to the absence of this I30 region.

Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein.

The small hydrophobic (SH) protein is a short channel-forming polypeptide encoded by the human respiratory syncytial virus (hRSV). Deletion of SH protein leads to the viral attenuation in mice and primates, and delayed apoptosis in infected cells. We have used a membrane-based yeast two-hybrid system (MbY2H) and a library from human lung cDNA to detect proteins that bind SH protein. This led to the identification of a membrane protein, B-cell associated protein 31 (BAP31). Transfected SH protein co-localizes with transfected BAP31 in cells, and pulls down endogenous BAP31. Titration of purified C-terminal endodomain of BAP31 against isotopically labeled SH protein in detergent micelles suggests direct interaction between the two proteins. Given the key role of BAP31 in protein trafficking and its critical involvement in pro- and anti-apoptotic pathways, this novel interaction may constitute a potential drug target.

X-linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis.

BACKGROUND: Mutation in the Wiskott-Aldrich syndrome Protein (WASP) causes Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN). The majority of missense mutations causing WAS and XLT are found in the WH1 (WASP Homology) domain of WASP, known to mediate interaction with WIP (WASP Interacting Protein) and CIB1 (Calcium and Integrin Binding). RESULTS: We analyzed two WASP missense mutants (L46P and A47D) causing XLT for their effects on T cell chemotaxis. Both mutants, WASPRL46P and WASPRA47D (S1-WASP shRNA resistant) expressed well in JurkatWASP-KD T cells (WASP knockdown), however expression of these two mutants did not rescue the chemotaxis defect of JurkatWASP-KD T cells towards SDF-1α. In addition JurkatWASP-KD T cells expressing these two WASP mutants were found to be defective in T cell polarization when stimulated with SDF-1α. WASP exists in a closed conformation in the presence of WIP, however both the mutants (WASPRL46P and WASPRA47D) were found to be in an open conformation as determined in the bi-molecular complementation assay. WASP protein undergoes proteolysis upon phosphorylation and this turnover of WASP is critical for T cell migration. Both the WASP mutants were found to be stable and have reduced tyrosine phosphorylation after stimulation with SDF-1α. CONCLUSION: Thus our data suggest that missense mutations WASPRL46P or WASPRA47D affect the activity of WASP in T cell chemotaxis probably by affecting the turnover of the protein.

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP.

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1(KD) cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1(KD) cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1(KD) cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1(KD) cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell-cell fusion. Toca-1(KD) cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP(KO) MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1(KD) cells restored myotube formation of Toca-1(KD) cells. Thus, our results suggest that Toca-1(KD) cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.

Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology.

Cerebrospinal fluid (CSF) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain. Defects in the ependymal layer functionality are a common cause of hydrocephalus. N-WASP (Neural-Wiskott Aldrich Syndrome Protein) is a brain-enriched regulator of actin cytoskeleton and N-WASP knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities. To shed light on the role of N-WASP in mouse brain development, we generated N-WASP conditional knockout mouse model N-WASP(fl/fl); Nestin-Cre (NKO-Nes). NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional N-WASP alleles. Importantly, all NKO-Nes mice developed cranial deformities due to excessive CSF accumulation and did not survive past weaning. Coronal brain sections of these animals revealed dilated lateral ventricles, defects in ciliogenesis, loss of ependymal layer integrity, reduced thickness of cerebral cortex and aqueductal stenosis. Immunostaining for N-cadherin suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls. Moreover, scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects. N-WASP deficiency does not lead to altered expression of N-WASP regulatory proteins, Fyn and Cdc42, which have been previously implicated in hydrocephalus pathology. Taken together, our results suggest that N-WASP plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus.

Wiskott-Aldrich Syndrome causing mutation, Pro373Ser restricts conformational changes essential for WASP activity in T-cells.

Wiskott-Aldrich Syndrome (WAS) is caused by mutations in Wiskott-Aldrich Syndrome Protein (WASP) and majority of the mutations are found in the WASP Homology 1 (WH1) domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the proline rich region (PRR) domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASPR(S339Y), WASPR(P373S)) or together (WASPR(SP/YS)) did not rescue the chemotaxis defect or membrane projection defect of Jurkat(WKD) T-cells (WASP knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP-WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASPR(S339Y) but not WASPR(P373S) or WASPR(SP/YS) rescued the IL-2 expression defect of Jurkat(WKD) T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASP(P373S) mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduces Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients.

Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT).

Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial-Mesenchymal Transition (EMT). EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM) probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor) is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor). Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation.

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.