We investigated fibrin-mediated gene transfer by embedding pDNA within the hydrogel during polymerization and using two modes of gene transfection with cells placed either on the surface (2D transfection) or within the hydrogel (3D transfection). Using this model, we found that cell transfection depended strongly on the local cell-pDNA microenvironment as defined by the 2D vs. 3D context, target cell type and density, as well as fibrinogen and pDNA concentrations. When cells were embedded within the fibrin matrix lipofectamine-induced cell death decreased significantly, especially at low target cell density. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner, suggesting that fibrin degradation maybe necessary for efficient gene transfer. We also provided proof-of-concept that fibrin-mediated gene transfer can be used for spatially localized gene delivery such as required in cell transfection microarrays. When lipoplexcontaining hydrogels were spotted in an array format gene transfer was strictly confined to pDNAcontaining fibrin spots with no cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may be used as a biomaterial to deliver genes in an efficient, cell-controlled and spatially-localized manner for potential applications in vitro or in vivo.

We recently reported that c-Jun N-terminal kinase (JNK) is associated with adherens junctions and phosphorylates β-catenin at serine 33/37 and threonine 41. Here, we report that inhibition of JNK led to formation of adherens junctions, which was accompanied by dissociation of α-catenin from the β-catenin/E-cadherin complex and increased association of α-catenin with the cytoskeleton. Conversely, activation of JNK increased binding of α-catenin to β-catenin, which was blocked by the JNK inhibitor SP600125 or JNK siRNA. In addition, inhibition of JNK failed to lead to adherens junction formation in cells where α-catenin was absent or knocked down. Conversely, introduction of α-catenin restored the responsiveness of cells to JNK inhibition and led to cell-cell adhesion. Experiments with domain deletion mutants showed that binding of α-catenin to β-catenin was required for transport of adherens junction complexes to the cell surface, while binding to actin was required for translocation to the cell-cell contact sites. Collectively, our results suggest that JNK affects the association of α-catenin with the adherens junction complex and regulates adherens junctions.

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We employed fibrin hydrogel as bioactive matrix for lentivirus mediated gene transfer. Fibrin-mediated gene transfer was highly efficient and exhibited strong dependence on fibrinogen concentration. Efficient gene transfer was achieved with fibrinogen concentration between 3.75 – 7.5 mg/mL. Lower fibrinogen concentrations resulted in diffusion of virus out of the gel while higher concentrations led to ineffective fibrin degradation by target cells. Addition of fibrinolytic inhibitors decreased gene transfer in a dose-dependent manner suggesting that fibrin degradation by target cells may be necessary for successful gene delivery. Under these conditions transduction may be limited only to cells interacting with the matrix thereby providing a method for spatially localized gene delivery. Indeed, when lentivirus-containing fibrin microgels were spotted in an array format gene transfer was confined to virus-containing fibrin spots with minimal cross-contamination between neighboring sites. Collectively, our data suggest that fibrin may provide an effective matrix for spatially-localized gene delivery with potential applications in high-throughput lentiviral microarrays and in regenerative medicine.

SummaryWe demonstrate that c-Jun N-terminal kinase (JNK) responds to substrate stiffness and regulates adherens junction (AJ) formation in epithelial cells in 2D cultures and in 3D tissues in vitro and in vivo. Rigid substrates led to JNK activation and AJ disassembly, whereas soft matrices suppressed JNK activity leading to AJ formation. Expression of constitutively active JNK (MKK7-JNK1) induced AJ dissolution even on soft substrates, whereas JNK knockdown (using shJNK) induced AJ formation even on hard substrates. In human epidermis, basal cells expressed phosphorylated JNK but lacked AJ, whereas suprabasal keratinocytes contained strong AJ but lacked phosphorylated JNK. AJ formation was significantly impaired even in the upper suprabasal layers of bioengineered epidermis when prepared with stiffer scaffold or keratinocytes expressing MKK7-JNK1. By contrast, shJNK1 or shJNK2 epidermis exhibited strong AJ even in the basal layer. The results with bioengineered epidermis were in full agreement with the epidermis of jnk1 2/2 or jnk2 2/2 mice. In conclusion, we propose that JNK mediates the effects of substrate stiffness on AJ formation in 2D and 3D contexts in vitro as well as in vivo.