Getting Autophagy to Akt: The protein kinase Akt is often activated in human cancers and is thought to promote tumor formation. One way in which it may do so is to inhibit autophagy (a process by which the cell digests its own proteins or organelles, especially damaged ones). Wang et al. (p. 956 , published online 25 October; see the Perspective by Koren and Kimchi ) provide a direct molecular mechanism by which Akt regulates autophagy. Beclin, a component of the autophagy machinery, appears to be a direct target of phosphorylation by Akt. Such phosphorylation enhanced interaction of Beclin with intermediate filaments of the cyto skeleton and inhibited autophagy. Expression of a modified Beclin 1 molecule that could not be phosphorylated by Akt inhibited Akt-induced transformation of cells in culture and tumor formation in a mouse model.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Summary Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and the S100A8 and S100A9 proteins, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1^−/− mice. IL-18 release was keratinocyte-autonomous, KRT1 and caspase-1 dependent, supporting an upstream role of KRT1 in the pathology. Finally, transcriptome profiling revealed a Krt1-mediated gene expression signature similar to atopic eczema and psoriasis, but different from Krt5 deficiency and epidermolysis bullosa simplex. Our data suggest a functional link between KRT1 and human inflammatory skin diseases.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10^−/−mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10^−/−mice suggests that there is a considerable redundancy in the keratin gene family.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.