Intersectin-2L Regulates Caveola Endocytosis Secondary to Cdc42-mediated Actin Polymerization

Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH_ITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH_ITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.The polymerization of actin has a central role in clathrin- and caveola-mediated endocytosis (1). Studies have shown a number of protein-protein interactions that suggest a functional relationship between the actin cytoskeleton and endocytic machinery; however, the underlying mechanisms remain unclear. ITSN-2L,² a multifunctional domain protein with two Epsin 15 homology domains, a central coiled-coil region followed by five consecutive Src homology 3 domains, a Dbl homology (DH), a pleckstrin homology (PH), and finally a C2 domain, interacts via the Src homology 3 region with the ubiquitously expressed neural Wiskott-Aldrich syndrome protein (N-WASP) that stimulates actin nucleation through Arp 2/3 complex activation (2). ITSN-2L interaction with N-WASP in turn induces activation of N-WASP in a Cdc42-dependent manner (2, 3). In this way, ITSN-2L on the basis of its DH domain acts as a GEF for the small Rho GTPase Cdc42, similar to its neuronal counterpart ITSN-1L (2, 4). The DH domain of ITSN-2L shows high sequence homology with the corresponding region of ITSN-1L (5), and it possesses all the amino acid residues required for its GEF enzymatic activity (6). Both long ITSN isoforms display immediately distal to the DH domain a PH domain, which may thereby modulate the intrinsic catalytic activity of the DH region (6–8). It has been shown that the PH domain enhances up to 100-fold the DH catalytic activity for some Dbl proteins compared with that measured for DH alone in vitro, whereas for other Dbl proteins the presence of the PH domain negatively regulates GEF activity of the DH region (9). This latter function is apparently mediated by interactions with phosphoinositides (7, 9). However, the PH sequence was shown to be dispensable for GEF activity of ITSN-2L in vitro, but it enhanced the ability to activate Cdc42 in vivo (9). Despite high sequence conservation among Rho GTPases, long ITSN isoforms apparently induce selective activation of Cdc42 due to the overall increasing size of the specificity residues of the GTPases (Cdc42 < Rac1 < RhoA) and the inability of ITSN to accommodate in an analogous position the larger size amino acid chains found in Rac1 and RhoA (10).ITSN-2L, like its alternatively spliced short isoform, is widely expressed in human tissue, and it shows subcellular distribution similar to components of the endocytic machinery (5). In COS-7 cells overexpressing ITSN-2 isoforms, clathrin-mediated transferrin uptake was blocked, consistent with their involvement in the regulation of clathrin-mediated endocytosis (5). By contrast, ITSN-2L overexpression in Jurkat cells stimulated T cell antigen receptor (TCR) internalization, whereas truncated ITSN-2L, deleted for the DH domain, caused significant inhibition of TCR internalization (2). The stimulatory effect of ITSN-2L on TCR endocytosis may be secondary to the ability of ITSN-2L to bind through its Src homology 3 domains the proline-rich domain of N-WASP followed by Cdc42-mediated actin polymerization (2). Although more work is needed to clarify these inconsistencies, both of these studies suggest that ITSN-2L may regulate endocytosis and function cooperatively with N-WASP and Cdc42 to link WASP-mediated actin polymerization to the endocytic machinery (2).Live cultured fibroblast imaging showed that actin polymerization as regulated by the WASP-Arp2/3 complex participates in the late stage of clathrin-mediated endocytosis (11). Therefore, we reasoned that ITSN-2L, as a specific activator of Cdc42, may be essential for actin cytoskeleton polymerization and caveola internalization in ECs. ECs are particularly rich in caveola, and caveola-mediated endocytosis is a fundamental step in mediating the transcytosis of proteins (12, 13), but the mechanisms of caveola-mediated endocytosis and the essential proteins involved remain enigmatic. In this study, we addressed the role of ITSN-2L in the mechanism of caveola internalization in ECs. Our data employing morphological, biochemical, and functional approaches show that ITSN-2L on the basis of Cdc42-mediated spatial actin polymerization is required in the mechanism of caveola-mediated endocytosis.     

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background

An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart.

Methodology/Principal Findings

Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ).

Conclusions/Significance

The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.     

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration,Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.     

Independent Association of HbA1c and Incident Cardiovascular Disease in People Without Diabetes

Recent studies have reported no association between elevated glycated hemoglobin (HbA_1c) and incident cardiovascular disease (CVD) among women without diabetes. This study describes associations between HbA_1c and new onset CVD in a representative adult population cohort. Assessment of participants in The North West Adelaide Health Study (NWAHS), a population study of randomly selected adults (age ≥18 years, n = 4,060), included measurement of height, weight, blood pressure, fasting lipids, glucose, and HbA_1c. A self‐completed questionnaire assessed doctor‐diagnosed diabetes, CVD and stroke, smoking status, and demographics. The cohort was followed for an average 3.5 years. Of the 2,913 adults free of diabetes at baseline and follow‐up, 94 (3.5%) reported new onset coronary heart disease (CHD) and/or stroke. Compared with those with an HbA_1c ≤5.0%, risk of new onset CVD was increased in those with HbA_1c 5.4–5.6% (odds ratio (OR) 2.5, 95% confidence interval (CI) 1.4, 4.6), and ≥5.7% (OR 1.9, 95% CI 1.1, 3.4), after adjustment for other risk factors. The association was stronger in women than men (P = 0.03), and attenuated to only a small degree by addition of impaired fasting glucose (IFG), hypertension, hypercholesterolemia, BMI, waist circumference, or smoking to the model. Elevated HbA_1c is related to new onset CVD over a relatively short follow‐up period in both men and women without diabetes and who do not develop diabetes, after adjustment for other major risk factors. Unlike previous studies, this relationship was not substantially attenuated by other traditional risk factors.     

Epigenetic Silencing of ��-Spectrin, a TGF-�� Signaling/Scaffolding Protein in a Human Cancer Stem Cell Disorder: BECKWITH-WIEDEMANN SYNDROME*

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology domain β-spectrin (β2SP) (the official name for human is Spectrin, beta, nonerythrocytic 1 (SPTBN1), isoform 2; the official name for mouse is Spectrin beta 2 (Spnb2), isoform 2), a scaffolding protein, functions as a potent TGF-β signaling member adaptor in tumor suppression and development. Yet, the role of the β2SP in human tumor syndromes remains unclear. Here, we report that β2SP^+/− mice are born with many phenotypic characteristics observed in BWS patients, suggesting that β2SP mutant mice phenocopy BWS, and β2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of β2SP is a new potential causal factor in human BWS patients. Furthermore, β2SP^+/− mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage.     

Characterization of peroxidase changes in resistant and susceptible warm-season turfgrasses challenged by <Emphasis Type="Italic">Blissus occiduus</Emphasis>

Peroxidases play an important role in plant stress related interactions. This research assessed the role of peroxidases in the defense response of resistant and susceptible buffalograsses [Buchloe dactyloides (Nutt.) Engelm] and zoysiagrasses (Zoysia japonica Steudel) to the western chinch bug, Blissus occiduus Barber. The objectives were: (1) to assess the relationships among protein content, basal peroxidase levels, chinch bug injury, and ploidy levels of chinch bug-resistant and -susceptible buffalograsses; (2) to compare peroxidase activity levels of resistant and susceptible buffalograsses and zoysiagrasses in response to chinch bug feeding; (3) and to analyze extracted proteins from chinch bug-resistant and -susceptible buffalograsses and zoysiagrasses by native gel electrophoresis to obtain information on the peroxidase profiles. Correlation analyses of 28 buffalograss genotypes with varying levels of chinch bug resistance and ploidy levels indicated that buffalograss total protein content was correlated (r = 0.47, P = 0.01) to chinch bug injury, while basal peroxidase levels was not (r = 0.19, P = 0.29), suggesting that the up-regulation of peroxidases in resistant buffalograsses is a direct response to chinch bug feeding. Three of the four chinch bug-resistant buffalograss genotypes evaluated had higher peroxidase activity in the infested plants compared to control plants. Peroxidase activity levels were similar between infested and control plants of the two highly susceptible buffalograss genotypes. Zoysiagrasses had lower peroxidase activity in general when compared to buffalograss control plants, and only ‘Zorro’ consistently showed higher peroxidase activity in the infested plants. Native gel electrophoresis analysis identified differences in the isozyme profiles of infested and control buffalograsses ‘Prestige’ and 196, and the zoysiagrass ‘Zorro’. Results from this study suggest that peroxidases have the potential to be used as markers for selecting chinch bug resistant turfgrasses, and may help explain how plants defend themselves against biotic stresses, such as chinch bugs.     

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.     

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background

Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified.

Methodology/Principal Findings

Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci.

Conclusions/Significance

This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.