Work during the current funding cycle implicates NF-kappaB/Rel proteins in keratinocyte-intrinsic growth control. In studying this, we found that the RelA NF-kappaB subunit controls the levels of the positive cell cycle regulator, CDK4. RelA physically interacts with CDK4, lowering CDK4 levels in a proteasome inhibitor-sensitive manner to trigger growth arrest. Conversely, RelA -/-epidermis generated recently in our lab displays increased CDK4 levels and hyper-proliferation. In human keratinocytes, NF-kappaB blockade increases CDK4 to levels sufficient to facilitate Ras-driven neoplasia. Using Cdk4-/- mice, we determined that epidermal hyperproliferation induced by loss of NF-kappaB function is CDK4-dependent. This increased proliferation is also dependent on the NF-kappaB-activating receptor, TNFR1, as shown by generating RelA-/-Tnfrl-/-epidermis. TNFR1 activates other signaling pathways besides IKK/NF-kappaB, notably the JNK cascade. In cells and tissue, NF-kappaB inhibition leads to JNK activation and JNK inhibitors block the hyper-proliferation that characterizes RelA -/- keratinocytes. In addition, RelA inhibits JNK activation, suggesting that epidermal RelA may oppose pro-proliferative signals from TNFR1 and JNK. Moreover, the majority of spontaneous human SCCs studied display evidence of simultaneous NF-kappaB blockade and JNK activation. These findings lead us to propose a model in which NF-kappaB RelA regulates epidermal proliferation in a manner involving the suppression of CDK4 protein levels and the opposition of the growth promoting action of TNFR1-JNK. First, we plan to define the mechanisms mediating CDK4 down regulation by NF-kappaB RelA in epidermal cells. To do this, we will define the nature and importance of the physical interactions between RelA and CDK4 as well as their impact on proteins involved in CDK4 stabilization and function. Second, we plan to characterize additional mechanisms involved in the increased epidermal proliferation induced by NF-kappaB inhibition. We will define the effects of altering the balance between JNK and NF-kappaB pathway function on epidermal homeostasis and neoplasia. Furthermore, we will define the contributions of TNFR1 to human epidermal neoplasia driven by NF-kappaB blockade. Over the proposed funding period, we plan to further define the mechanisms through which NFkappaB regulates growth of both normal and neoplastic epidermal tissue.
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