ZO-1: Lamellipodial Localization in a Corneal Fibroblast Wound Model

PURPOSE. To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS. Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to ␤-catenin, cadherins, connexins, integrins, ␣-actinin, and cortactin were used. G-and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS. As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, ␣-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins ␣5␤1 and ␣v␤3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS. ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization. I n the stroma of the normal cornea, keratocytes communicate with one another by gap junctions. 1-3 Wounding disrupts these junctions, and, during repair, corneal fibroblasts and myofibroblasts reestablish this communication. 4 -6 Using an in vitro corneal stromal cell wound-healing model, we have demonstrated that these connections are functional gap junctions. We have also identified cadherin-based intercellular junctions in fibroblasts and myofibroblasts. 9,10 Furthermore, ZO-1 has been shown to associate directly with connexins, facilitating their lateral aggregation before intercellular junction formation, and regulating their removal from the cell surface. 10,11 ZO-1 is a 220-kDa cytoplasmic protein that was first described as a component of tight junctions (zonula occludens) of epithelial and endothelial cells. From the Departments of 1 Ophthalmology an

Sproutv1, a wt1 target gene, anagonizes the action of, receptor tyrosine kinases

WT1 is a tumor suppressor gene with an essential role in the development of the kidney. In addition WT1 in expressed in hematopoietic progenitor cells and can inhibit hematopoietic cell growth. In a model system for WT1 action we found that in addition to inhibiting cell growth.WTl could induce features of epithelial differentiation in NIH 3T3 fibroblasts. Using this cell line model we performed representative difference analysis to isolate genes regulated by WT1. The genes isolated were then tested for their ability to be induced after transient transduction of naive 3T3 cells with a WT1-harboring retrovirus, their expression in a podocyte and mesonephric cell line and finally for their expression in the developing murine kidney. Among the genes meeting all these criteria was sprouty 1. Sprouty was originally isolated in Drosophila as an inhibitor of FGF signaling and branching morphogensis of the respiratory system of the fly. In order to understand the potential function of sprouty in organogenesis and tumor development, we established cells that conditionally expressed sprouty. Sproutyl as well as sprouty 2 inhibited growth of NIH 3T3 cells. Sprouty inhibited the activation of ERK by FGF and PDGF and inhibited the ability of growth factors to activate the ELK1, API and NFkB transcription factors. Inhibition of NFkB activity was specific to the PDGF and not the TNF pathway. While the RAS/RAF/MEK pathway was inhibited by sprouty, activation of AKT through the PI3 kinase pathway was not, again indicating a specific point of action of sprouty downstream of the receptor tyrosine kinase. In this regard, sprouty inhibited the activation of ERK, MEK and inhibited the binding of RAS to RAF. This suggest that sprouty either prevents the accumulation of GTP-RAS or inhibts the RAS/RAF interaction. These data indicate that sprouty is growth inhibitor downstream of WT1 whose expression in response to WT1 and growth factor signaling may limit proliferation during organ development and hematopoiesis

Regulation of JNK signaling by GSTp

Studies of low basal Jun N-terminal kinase (JNK) activity in non-stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S-transferase Pi (GSTp) and was characterized as a JNK-associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp-JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun-JNK complex caused a dose-dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp-derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c-Jun ubiquitination and decreased c-Jun-mediated transcription. Co-transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1-MKK4 module. Mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases

Fluorine-labeled Dasatinib Nanoformulations as Targeted Molecular Imaging Probes in a PDGFB-driven Murine Glioblastoma Model

Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR) inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380), in combination with nanocarrier vehicles and metabolic imaging tools (microPET) to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB)-driven genetically engineered mouse model (GEMM) of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using 18F-SKI249380 and 18F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg) versus saline vehicle (P < .001) in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for 18F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g); P = .002] compared to control values (1.6%ID/g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g), consistent with in vivo binding specificity. Using 18F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24–60 nM) that were comparable to in vitro 50% inhibitory concentration values. 18F-SKI249380 is a PDGFR-selective tracer, which demonstrates improved delivery to PDGFB-driven high-grade gliomas and facilitates treatment planning when coupled with nanoformulations and quantitative PET imaging approaches

Multimodal silica nanoparticles are effective cancer-targeted probes in a model of human melanoma

Nanoparticle-based materials, such as drug delivery vehicles and diagnostic probes, currently under evaluation in oncology clinical trials are largely not tumor selective. To be clinically successful, the next generation of nanoparticle agents should be tumor selective, nontoxic, and exhibit favorable targeting and clearance profiles. Developing probes meeting these criteria is challenging, requiring comprehensive in vivo evaluations. Here, we describe our full characterization of an approximately 7-nm diameter multimodal silica nanoparticle, exhibiting what we believe to be a unique combination of structural, optical, and biological properties. This ultrasmall cancer-selective silica particle was recently approved for a first-in-human clinical trial. Optimized for efficient renal clearance, it concurrently achieved specific tumor targeting. Dye-encapsulating particles, surface functionalized with cyclic arginine–glycine–aspartic acid peptide ligands and radioiodine, exhibited high-affinity/avidity binding, favorable tumor-to-blood residence time ratios, and enhanced tumor-selective accumulation in αvβ3 integrin–expressing melanoma xenografts in mice. Further, the sensitive, real-time detection and imaging of lymphatic drainage patterns, particle clearance rates, nodal metastases, and differential tumor burden in a large-animal model of melanoma highlighted the distinct potential advantage of this multimodal platform for staging metastatic disease in the clinical setting