Localization of ZO-1 in the nucleolus of corneal fibroblasts.

PURPOSE: Within the multidomain structure of ZO-1 are motifs responsible for ZO-1's localization to intercellular junctions and its newly demonstrated localization to the leading edge of lamellipodia in corneal fibroblasts. Since ZO-1 also has two nuclear localization signals, this study was undertaken to determine whether stimuli associated with wounding would induce nuclear translocation of ZO-1 METHODS: Immunocytochemistry and immunoblot analysis were used to localize endogenous and exogenous ZO-1 in nuclear and cytoplasmic sites in corneal fibroblasts and 293T fibroblasts, with and without myc-ZO-1 transfection. Cells were serum starved by growth for 48 hours in DMEM/F12 with 0.2% FBS and subsequently were either scrape wounded or treated with 10% FBS, PDGF, or FGF-2 for 6 hours. For immunoblot analysis, after lysis, the nuclear and cytosolic fractions were separated and analyzed by SDS-PAGE. Cells on companion coverslips were fixed with 3% p-formaldehyde and permeabilized with 1% Triton before immunocytochemical detection of ZO-1 and nuclear proteins. RESULTS: ZO-1 was rarely detected in the nucleus of serum-starved corneal fibroblasts. In contrast, it colocalized with nucleolin in the nucleoli of corneal fibroblasts after serum-starved cells were treated with 10% FBS, PDGF, or FGF-2. Immunoblot analysis confirmed the immunocytochemical results: Little ZO-1 was detected in the nuclear fraction of lysates of serum-starved cells, but ZO-1 was found in the nuclear fractions of rabbit corneal and 293T fibroblasts treated with 10% FBS, PDGF, or FGF-2. Furthermore in scrape-wounded corneal fibroblasts, ZO-1 was localized to nucleoli of both serum-starved and serum-treated cells. CONCLUSIONS: Localization of ZO-1 to nucleoli of corneal and 293T fibroblasts under proliferative and promigratory conditions suggests a physiologically significant interaction of ZO-1 with proteins in nucleoli during the healing process.

FAK-dependent regulation of myofibroblast differentiation.

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFbeta) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGFbeta induces expression of smooth muscle alpha actin (SMalphaA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFbeta induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGFbeta-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFbeta-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK -/- MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMalphaA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK -/- [corrected]MEFs. We conclude that FAK does not contribute to TGFbeta-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SMalphaA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.

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 beta-catenin, cadherins, connexins, integrins, alpha-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, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 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.

Urokinase anchors uPAR to the actin cytoskeleton.

PURPOSE: To investigate the expression and localization of urokinase plasminogen activator (uPA) and its receptor (uPAR) and their interaction with the actin cytoskeleton in human corneal fibroblasts. METHODS: Primary cultured human corneal fibroblasts were exposed to exogenous uPA to investigate its effect on the distribution of uPAR under resting conditions and in a scrape-wound model. Fluorescence microscopy, immunolocalization, immunoprecipitation, and the actin depolymerizing drug cytochalasin D were used to evaluate uPAR's interaction with the actin cytoskeleton. RESULTS: uPA/uPAR was immunodetected in large (200 microm2) aggregates devoid of detectable F-actin. However, when uPA was added to corneal fibroblasts before fixation, a dynamic association between uPAR and the actin cytoskeleton was revealed: the uPA/uPAR complex was immunodetected throughout the surface of the plasma membrane in the form of dispersed small aggregates (0.05 microm2). Association of uPAR with actin stress fibers was visualized when FITC-labeled uPA was added to the cells. This codistribution of uPA/uPAR and actin was not detected when the cells were pretreated with the actin-depolymerizing drug, cytochalasin D. uPAR was found also in focal adhesions, the termination points of F-actin, where it colocalized with the integrin alphavbeta3 in cells migrating into a scrape wound. Coimmunoprecipitation experiments confirmed the physical association of uPAR with alphavbeta3 in fibroblasts. CONCLUSIONS: The authors propose that uPA/uPAR ligation anchors the complex to the actin cytoskeleton and is a part of the mechanism responsible for uPA-induced cell migration in fibroblasts.