Dysregulated ΔNp63α negatively regulates the maspin promoter in keratinocytes via blocking endogenous p73 binding

Abstract

While overexpression of the p63 isoform, ΔNp63α, has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed ΔNp63α aberrantly maintains proliferation of primary mouse keratinocytes under conditions that normally induce growth arrest and differentiation. To identify genes downstream of dysregulated ΔNp63α that may contribute to squamous cancer development and progression, we performed microarray analyses using primary mouse keratinocytes. Herein we report that elevated ΔNp63α differentially regulates genes involved in a variety of cellular functions. Of note, multiple protease inhibitor mRNAs were downregulated including: maspin (serpinB5); plasminogen activator inhibitor-2 (PAI-2; serpinB2); and tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted TIMP-3 and PAI-2 protein declined in the presence of dysregulated ΔNp63α, however secreted maspin remained stable. Intracellular maspin protein expression decreased in response to overexpressed ΔNp63α, as did PAI-2. In contrast, TIMP-3 protein was not detected intracellularly, supporting a solely extracellular function. Electrophoretic mobility shift assays (EMSAs) using a maspin promoter p53/p63 consensus sequence revealed endogenous transcription factor(s) binding to this sequence in keratinocytes that was disrupted by overexpressed ΔNp63α. This was confirmed by ChIP assays. This binding was interrupted by the addition of antibodies recognizing p73, but not p53 or p63, and significantly diminished in EMSA reactions from p73(-/-) keratinocytes, confirming p73 as a constituent. Physical association between p73/ΔNp63α was observed in control β-gal overexpressing keratinocytes and was enhanced in the presence of overexpressed ΔNp63α These findings underscore the importance of properly balanced p53 homologs for tissue homeostasis.

Keywords: p53 homologs; protease inhibitory proteins; squamous cell cancer.