p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to down-regulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibited hypoproliferation, differentiation defects were not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescued the cell proliferation defect of p63 knockdown alone but failed to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Furthermore, ⌬Np63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes.[Keywords: Epidermis; skin; differentiation; proliferation; p63; p53] Supplemental material is available at http://www.genesdev.org.

Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.Supplemental material is available at http://www.genesdev.org.Received August 29, 2007; revised version accepted December 3, 2007. Epithelial tissues such as skin demonstrate remarkable anatomic diversity in their structure and function. For instance, while long terminal hairs decorate the scalp, palmo-plantar skin lacks hairs but possesses thickened epidermal barriers for mechanical stress. These anatomic differences lead to many body site-specific manifestations of diseases and guide their appropriate treatments (Bolognia et al. 2003). Because the epidermis is continually turned over, the apparent stability of sitespecific features raises the fundamental question of how positional identity is acquired and maintained in the skin. Classic heterotypic recombination experiments in chick suggested that a primary dermal signal may dictate the positional identity of epithelial differentiation (Dhouailly 1984). We and others have used a genomic approach to examine the anatomic diversity of gene expression of cell types in the dermis and identified fibroblasts as a cell type that demonstrates consistent and large-scale differences of the expression of cell-cell signaling proteins in a site-specific manner (Chang et al. 2002;Chi et al. 2003;Rinn et al. 2006). Systematic comparison of the global transcriptional programs of fibroblasts from anatomic sites that finely map the body suggest that fibroblasts are differentiated based on their position along three anatomic divisions: anterior-posterior, proximal-distal, and dermal-nondermal (Rinn et al. 2006). Moreover, primary adult fibroblasts retained many features of the embryonic pattern of expression of HOX genes (Chang et al. 2002;Rinn et al. 2006), a family of homeodomain transcription factors that act to specify positional identity during development. These data support the idea, first proposed by Chuong (1993), that a "HOX code" may dictate the positional identity of skin and thus influence site-specific epidermal differentiation. However, fundamental elements of this hypothesis, such as the stability of HOX expression, gene regulatory function, and inductive activities governed by HOX genes in adult fibroblasts remain poorly understood. Here we show that expression of HOXA13 is required and the transcriptio...

SUMMARY In the absence of low-level endoplasmic reticulum-to-mitochondrial Ca2+ transfer, ATP levels fall and AMPK-dependent, mTOR-independent autophagy is induced as an essential survival mechanism in many cell types. Here we demonstrate that tumorigenic cancer cell lines, transformed primary human fibroblasts and tumors in vivo respond similarly, but autophagy is insufficient for survival, and cancer cells die while their normal counterparts are spared. Cancer cell death is due to compromised bioenergetics that can be rescued with metabolic substrates or nucleotides, and caused by necrosis associated with mitotic catastrophe during their proliferation. Our findings reveal an unexpected dependency on constitutive Ca2+ transfer to mitochondria for viability of tumorigenic cells and suggest that mitochondrial Ca2+ addiction is a feature of cancer cells.

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces "SASP-like" inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS-or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.

Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin. Lysosomal activity was required for mechanistic target of rapamycin signaling and mitochondrial oxidative metabolism. In turn, mitochondrial reactive oxygen species, produced as a natural byproduct of oxidative phosphorylation, were necessary for keratinocyte differentiation. Finally, treatment with exogenous reactive oxygen species rescued the differentiation defect in lysosome-inhibited keratinocytes. These findings highlight a reciprocal relationship between lysosomes and mitochondria, in which lysosomes support mitochondrial metabolism and the associated production of mitochondrial reactive oxygen species. The mitochondrial reactive oxygen species released to the cytoplasm in suprabasal keratinocytes triggers autophagy and lysosome-mediated degradation necessary for epidermal differentiation. As defective lysosome-dependent autophagy is associated with common skin diseases including psoriasis and atopic dermatitis, a better understanding of the role of lysosomes in epidermal homeostasis may guide future therapeutic strategies.