Netherton syndrome (NS) is a human autosomal recessive skin disease caused by mutations in the SPINK5 gene, which encodes the putative proteinase inhibitor LEKTI. We have generated a transgenic mouse line with an insertional mutation that inactivated the mouse SPINK5 ortholog. Mutant mice exhibit fragile stratum corneum and perinatal death due to dehydration. Our analysis suggests that the phenotype is a consequence of desmosomal fragility associated with premature proteolysis of corneodesmosin, an extracellular desmosomal component. Our mouse mutant provides a model system for molecular studies of desmosomal stability and keratinocyte adhesion, and for designing therapeutic strategies to treat NS.Supplemental material is available at http://www.genesdev.org.Received June 17, 2004; revised version accepted August 6, 2004. Netherton Syndrome (NS) (MIM 256500) is a severe, recessively inherited skin disease in humans with high neonatal lethality. It is characterized by ichthyosiform erythroderma, atopic dermatitis, bamboo hair, skin barrier defects, and elevated IgE levels in survivors (Krafchik and Toole 1983;Judge et al. 1994). Mutations have been identified in the SPINK5 (serine proteinase inhibitor Kazal type 5) gene of NS patients (Chavanas et al. 2000;Sprecher et al. 2001;Walley et al. 2001;Bitoun et al. 2002;Komatsu et al. 2002). SPINK5 encodes LEKTI (lympho-epithelial Kazal-type-related inhibitor), which is a putative proteinase inhibitor that contains an N-terminal signal peptide and 15 domains with high internal homology (Magert et al. 1999). Each domain has four conserved cysteines. Domains 2 and 15 possess two additional cysteines, which make them typical Kazal-type proteinase inhibitor domains (Magert et al. 1999). LEKTI exhibits proteinase inhibitor activity in vitro (Magert et al. 1999;Komatsu et al. 2002;Walden et al. 2002;Mitsudo et al. 2003). In NS patients, loss or reduction of LEKTI activity is presumed to result in elevated proteolytic activity in the suprabasal epidermis, leading to erythroderma and skin-barrier defects. However, the specific proteins that are targeted for degradation in these patients have not been identified. We describe here a Spink5 mutant mouse line that shows severe skin defects associated with desmosomal fragility, and thus, provides insights into the molecular pathogenesis of NS and a novel model system for studies of keratinocyte adhesion. Results and DiscussionTransgenic mouse line OVE1498 was generated by coinjection of a tyrosinase-tagged (Yokoyama et al. 1990) Sleeping Beauty transposon (Ivics et al. 1997) (termed pT-Tybs-3ЈE) along with PGK2-SB10 (Ivics et al. 1997) (see Supplementary Fig. S1) into inbred FVB/N embryos. The transgenic founder and its transgenic F1 offspring were phenotypically normal and showed no evidence for transposition of the transgene(s) (data not shown). When transgenic F1 mice were intercrossed, ∼25% of the newborn offspring developed severe skin blistering and water barrier defects, leading to death within several hours after birth ...

Triple negative breast cancer (TNBC), characterized by an abundance of treatment-resistant breast cancer stem cells (CSCs), has a poorer prognosis than other types of breast cancers. Despite its aggressiveness, no effective targeted therapy exists for TNBC. Here, we demonstrate that CQ effectively targets CSCs via autophagy inhibition, mitochondrial structural damage, and impairment of double-stranded DNA break repair. Electron microscopy demonstrates CQ-induced mitochondrial cristae damage, which leads to mitochondrial membrane depolarization with a significant reduction in the activity of cytochrome c oxidase and accumulation of superoxide and double-stranded DNA breaks. CQ effectively diminishes the TNBC cells’ ability to metastasize in vitro and in a TNBC xenograft model. When administered in combination with carboplatin, CQ effectively inhibits carboplatin-induced autophagy. This combination treatment significantly diminishes the expression of DNA repair proteins in CSC subpopulations, resulting in tumor growth reduction in carboplatin-resistant BRCA1 wild-type TNBC orthotopic xenografts. As TNBC’s high treatment failure rate has been attributed to enrichment of CSCs, CQ, an autophagy inhibitor with anti-CSC effects, may be an effective adjunct to current TNBC chemotherapy regimens with carboplatin.

Although some of the transcription factors that are required for expression of crystallins during lens development have been identified, the molecular interactions that contribute to enhanced crystallin expression are not yet well defined. In this study, we designed experiments to test whether the co-activators CREB-binding protein (CBP) and/or p300 interact with c-Maf, Prox-1, or Sox-1 to enhance transcription of crystallin genes. Promoter regions from the mouse ␣A-, ␤B2-, and ␥F-crystallin genes were linked to a luciferase reporter. Expression of c-Maf transactivated each of these promoters. Of particular interest, co-expression of CBP or p300 with c-Maf was found to synergistically co-activate each promoter. CBP and p300 were less effective or ineffective at co-activation with Prox-1 or Sox-1. Co-immunoprecipitation and mammalian two-hybrid experiments revealed that CBP and p300 bind to c-Maf and Prox-1 but not to Sox-1. The co-activation of c-Maf by CBP/p300 requires histone acetyltransferase activity. Our results suggest that c-Maf recruits CBP and/or p300 to crystallin promoters leading to up-regulation of crystallin gene expression through localized histone acetylation and consequent chromatin re-modeling. In a promoter-specific fashion, co-activation can be modulated by Prox-1 and/or Sox-1. This modulation may help to specify the endogenous levels of crystallin gene expression.

Break-induced replication (BIR) is a DNA double-strand break repair pathway that leads to genomic instabilities similar to those observed in cancer. BIR proceeds by a migrating bubble where asynchrony between leading and lagging strand synthesis leads to accumulation of long single-stranded DNA (ssDNA). It remains unknown how this ssDNA is prevented from unscheduled pairing with the template, which can lead to genomic instability. Here, we propose that uncontrolled Rad51 binding to this ssDNA promotes formation of toxic joint molecules that are counteracted by Srs2. First, Srs2 dislodges Rad51 from ssDNA preventing promiscuous strand invasions. Second, it dismantles toxic intermediates that have already formed. Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules. Overall, we uncover a new feature of BIR and propose that tight control of ssDNA accumulated during this process is essential to prevent its channeling into toxic structures threatening cell viability.