[PubMed] [Google Scholar]Zhang G., Njauw C. further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral PF-06700841 P-Tosylate expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1Cbased cues from their neighbors to facilitate entry into a terminal differentiation pathway. INTRODUCTION Eph receptors comprise the largest family of mammalian receptor tyrosine kinases that mediate cellCcell communication by interacting with ephrin ligands on adjacent cells (Miao and Wang, 2009 PF-06700841 P-Tosylate ; Pasquale, 2010 ). The Eph receptors are subdivided into A and B subfamily members depending on their binding preference for glycosylphosphatidylinositol (GPI)-linked ephrin-A or transmembrane ephrin-B ligands. Interestingly, Eph/ephrin complexes signal in a bidirectional manner with forward pathways emanating through the receptor and reverse signaling activated via the ephrins. A variety of Eph receptors and ephrin ligands are expressed during embryogenesis, where they play critical and often overlapping roles in brain and blood vessel development. Eph/ephrin expression is altered in many tumors and leads PF-06700841 P-Tosylate to changes in cancer cell adhesion, cytoskeleton, migration, proliferation, and survival. Eph receptors and ephrins are also present in adult epithelial tissues, where their roles are beginning to be elucidated. EphA2 was originally cloned from a human epithelial (HeLa) cDNA library (Lindberg and Hunter, 1990 ). The expression and activation of EphA2 in epithelial cells is tightly regulated by the calcium-dependent cell adhesion molecule, E-cadherin (Zantek null mutant mouse keratinocytes (Guo (2008) . Keratinocytes were incubated in the PF-06700841 P-Tosylate presence of recombinant mouse ephrin-A1-Fc, ephrin-B1-Fc (R&D Systems, Minneapolis, MN) or human Fc (Jackson Labs) as a control. The dimeric peptides were added directly to the culture medium or after preclustering with an anti-human Fc IgG (1:5 ratio of antibody to peptide) for 30 min as described previously (Shi exfoliative toxin A (ETA) was provided by John Stanley (University of Pennsylvania) and generated as previously described (Getsios for 5 min at 4C to remove PBS/ethanol and resuspended in propidium iodide staining solution [0.1% Triton-X100, 50 g/ml propidium iodide (Sigma-Aldrich), and 0.2 mg/ml RNaseA in PBS], vortexed, and incubated for 20 min at 37C. For some experiments, cultures were treated with 10 M U0126 (Cell Signaling Technology) for the duration of the calcium switch. To assess colony growth capacity, a crystal violet assay was performed on keratinocytes plated at 1.5 103 cells/cm2 in a six-well plate, switched into 0.03 or 1.2 mM calcium, and treated with Fc or ephrin-A1-Fc peptides every 48 h. Cultures were harvested 7 d after ligand treatment. Plates were washed gently at room temperature with PBS and then incubated with 0.5% crystal violet (Sigma-Aldrich) in a 20% methanol solution for 15 min at room temperature. Plates were then washed twice in water, and colony images were acquired on a Leica MZ6 dissecting stereomicroscope with a Hamamatsu PF-06700841 P-Tosylate Orca digital camera (model C4742-95) using MetaVue software (Universal Imaging) and analyzed using ImageJ software. Particle analysis was performed on duplicate wells for each condition to obtain the surface area (in pixels2) of resultant keratinocyte colonies. The watershed function was used on each image to separate colonies that overlapped. Dispase-Based Mechanical Dissociation Assay Keratinocytes were seeded to confluency (3 105 cells/cm2) for 24 h in M154 containing 0.07 mM calcium and switched into 0.4 mM Rabbit polyclonal to IkB-alpha.NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA (MIM 164014), or RELB (MIM 604758) to form the NFKB complex.The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm. calcium containing Fc or ephrin-A1-Fc peptides for an additional 48 h or 1.2 mM calcium for 2 h. In some experiments, 2.0 g/ml ETA was added 2 h before the addition of dispase to cleave the extracellular domain of desmoglein 1 as previously described (Getsios (2009) . Data are represented as the average number.