Adherens junctions in epithelial cells and in bioengineered tissues
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Recently it was demonstrated that JNK acts as a switch, signaling adherens junction formation. Inhibition of JNK chemically by SP600125 or by using a dominant negative JNK system promotes the formation of adherens junction, in vitro . It was also shown that &agr;-catenin leaves the E-cadherin/β-catenin complex and binds to actin and reorganizes the cytoskeleton, upon junction formation. However, in some cell types it was observed that this separation was not required although JNK inhibition resulted in adherens junctions formation as observed in keratinocytes. In order to test if this separation is necessary for junction formation, &agr;-catenin was mutated in such a way that its interaction with β-catenin was enhanced and it did not separate from β-catenin upon JNK inhibition. It was seen that junctions were still formed suggesting that the separation is not required for adherens junctions formation. However, it may be interesting to know if this separation is required for actin reorganization as &agr;-catenin cannot bind actin and β-catenin simultaneously. Preliminary data have shown negative correlation between p-JNK levels and adherent junction formation in human foreskin tissue. Here, we demonstrate that by genetic modification of JNK activity, the pattern of adherent junction formation can be changed in vivo and in bioengineering skin equivalents. In order to recapitulate the physiological conditions in-vitro, we constructed tissue engineered skin equivalents to mimic the human epidermis. Further, we also engineered skin made of siRNA JNK1 and siRNA JNK2 knockdown keratinocytes. JNK knockdown affects proliferation and cell adhesion in the tissue. The skin equivalents made from the siRNA JNK1 cells have lesser number of layers and siRNA JNK2 cells have more layers than the wild type. Also, these skins made from knockdown cells have a significantly higher intensity of adherens junctions even in the basal layer compared to the wild type epidermis. We confirmed these results in vivo , in skin samples taken from genetically modified mouse carrying the JNK knockout genes. Similar results were seen in the proliferation and adherens junctions of the mouse skin, in accordance with bioengineered skin data. These results show that inhibition of JNK activity is required for adherens junction formation in a three dimensional setting as well. Since JNK has been associated with certain tumors, the effect of JNK on adherens junction formation may help us understand tumor progression better.