p63 is a recently identified homolog of p53 that is found in the basal layer of several stratified epithelial tissues such as the epidermis, oral mucosa, prostate, and urogenital tract. Studies with p63 ؊/؊ mice and analysis of several human autosomal-dominant disorders with germ line p63 mutations suggest p63 involvement in maintaining epidermal stem cell populations. The p63 gene encodes six splice variants with reported transactivating or dominant-negative activities. The goals of the current study were to determine the splice variants that are expressed in primary human epidermal keratinocytes (HEKs) and the biochemical activity p63 has in these epithelial cell populations. We found that the predominant splice variant expressed in HEKs was ⌬Np63␣, and it was present as a phosphorylated protein. During HEK differentiation, ⌬Np63␣ and p53 levels decreased, while expression of p53 target genes p21 and 14-3-3 increased. ⌬Np63␣ had transcriptional repressor activity in vitro, and this activity was reduced in ⌬Np63␣ proteins containing point mutations, corresponding to those found in patients with Hay-Wells syndrome. Further, we show that ⌬Np63␣ and p53 can bind the p21 and 14-3-3 promoters in vitro and in vivo, with decreased binding of p63 to these promoters during HEK differentiation. These data suggest that ⌬Np63␣ acts as a transcriptional repressor at select growth regulatory gene promoters in HEKs, and this repression likely plays an important role in the proliferative capacity of basal keratinocytes.

Summary We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus, hippocampal dysgenesis, sterility and chronic inflammation/infection of lung, middle ear and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells, and suggest that p73 ‘marks’ these cells for MCC differentiation. In sum, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation and, like p63, has an essential role in development of tissues.

There is increasing evidence that prolonged mitotic arrest initiates apoptosis; however, little is known about the signaling pathways involved. Several studies have associated deregulated Cdc2 activity with apoptosis. Herein, we report that the anti-apoptotic protein, Bcl-2, undergoes cell cycle-dependent phosphorylation during mitosis when there is elevated Cdc2 activity. We found that paclitaxel (Taxol ® ) treatment of epithelial tumor cells induced a prolonged mitotic arrest, elevated levels of mitotic kinase activity, hyperphosphorylation of Bcl-2, and subsequent cell death. The Taxol-induced Bcl-2 phosphorylation was dose-dependent. Furthermore, phosphorylated Bcl-2 remained complexed with Bax in Taxol-treated cells undergoing apoptosis. Immunoprecipitation experiments revealed a Bcl-2-associated kinase capable of phosphorylating histone H1 in vitro. However, the kinase was likely not cyclin B1/Cdc2, since cyclin B1/Cdc2 was not detectable in Bcl-2 immunoprecipitates, nor was recombinant Bcl-2 phosphorylated in vitro by cyclin B1/Cdc2. The results of this study further define a link between mitotic kinase activation and the apoptotic machinery in the cell. However, the role, if any, of prolonged Bcl-2 phosphorylation in Taxol-mediated apoptosis awaits further definition of Bcl-2 mechanism of action. Taxol may increase cellular susceptibility to apoptosis by amplifying the normal downstream events associated with mitotic kinase activation.

Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21 Waf1 , MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21 Waf1 , MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.

The cloned Kv1.5 K+ channel displays similar kinetics and pharmacology to a delayed rectifier channel found in atrial myocytes. To determine whether the Kv1.5 isoform plays a role in the cardiac action potential, it is necessary to confirm the expression of this channel in cardiac myocytes. Using antibodies directed against two distinct channel epitopes, the Kv1.5 isoform was localized in human atrium and ventricle. Kv1.5 was highly localized at intercalated disk regions as determined by colocalization with connexin and N-cadherin specific antibodies. While both antichannel antibodies localized the Kv1.5 protein in cardiac myocytes, only the NH2-terminal antibodies stained vascular smooth muscle. The selective staining of vasculature by this antiserum suggests that epitope accessibility, and perhaps channel structure, varies between cardiac and vascular myocytes. Kv1.5 expression was localized less in newborn tissue, with punctate antibody staining dispersed on the myocyte surface. This increasing organization with age was similar to that observed for connexin. Future work will address whether altered K+ channel localization is associated with cardiac disease in addition to changing with development. (J. Clin. Invest. 1995. 96:282-292.)