Michael Devos

Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H2O2-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H2O2-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A2 activities, whereas H2O2-induced necrosis requires iron-dependent Fenton reactions.

Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing Factors in the Skin

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.

Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20EKO) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20EKO mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

Cell-Type-Specific Chromatin States Differentially Prime Squamous Cell Carcinoma Tumor-Initiating Cells for Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) in cancer cells has been associated with metastasis, stemness, and resistance to therapy. Some tumors undergo EMT while others do not, which may reflect intrinsic properties of their cell of origin. However, this possibility is largely unexplored. By targeting the same oncogenic mutations to discrete skin compartments, we show that cell-type-specific chromatin and transcriptional states differentially prime tumors to EMT. Squamous cell carcinomas (SCCs) derived from interfollicular epidermis (IFE) are generally well differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT, efficiently form secondary tumors, and possess increased metastatic potential. Transcriptional and epigenomic profiling revealed that IFE and HF tumor-initiating cells possess distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT that correlate with accessibility of key epithelial and EMT transcription factor binding sites. These findings highlight the importance of chromatin states and transcriptional priming in dictating tumor phenotypes and EMT.

Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation.

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.

Caspase‐14 overexpression in hairless mice is not involved in utricle formation

Loss of functional hairless (HR) transcriptional repressor leads to utricle formation and congenital hair loss both in mice and men. Studies in mice have shown that this is preceded by overexpression of caspase‐14 at the infundibulum in the hair follicle before conversion to utricle occurs. In this report, we show that HR regulates caspase‐14 expression dependent on its interaction with histone deacetylases, implicating chromatin remodelling in the transcriptional regulation of caspase‐14. However, crossing hairless mutant mice with caspase‐14‐deficient mice revealed that caspase‐14 overexpression is not the cause of utricle formation.

Keratinocyte Expression of A20/TNFAIP3 Controls Skin Inflammation Associated with Atopic Dermatitis and Psoriasis

Keratinocytes are key players in chronic inflammatory skin diseases. A20 regulates NF-κB-dependent expression of proinflammatory genes and cell death, but the impact of its expression in keratinocytes on systemic inflammation and skin disorders has not been determined. Comparative transcriptomic analysis of microdissected epidermis showed that A20 is down-regulated in involved epidermis, but not in dermis, of psoriasis and atopic dermatitis patients, suggesting that loss of A20 expression in keratinocytes increases the vulnerability for psoriasis/atopic dermatitis induction. We have previously shown that epidermis-specific A20 knockout mice (A20EKO) develop mild epidermal hyperplasia but no macroscopic skin inflammation. We now show that various cytokines and chemokines are up-regulated in A20EKO mouse skin. A20EKO mice also display systemic proinflammatory changes, even in the absence of skin immune cell infiltration, and an exacerbated disease severity upon induction of experimental psoriasis, atopic dermatitis, or skin barrier disruption. Keratinocytes showed increased proinflammatory gene expression in the absence of A20 in unstimulated and IL-17A-stimulated conditions, in part resulting from uncontrolled MyD88-dependent signaling. Our findings indicate that absence of A20 in keratinocytes leads to systemic inflammation at homeostatic conditions and is sufficient to exacerbate inflammatory skin disorders associated with different immune profiles by increasing cytokine and chemokine expression.

ΔNp63α Acts as a Lineage-Survival Oncogene in Squamous Cell Carcinoma

Michael Devos1,2 and Wim Declercq1,2* 1Molecular Signaling and Cell Death Unit, VIB Center for Inflammation Research, Ghent, Belgium 2Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium *Corresponding author: Wim Declercq, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium, Tel: +32-(0)9 33 13721; Fax: +32-(0)9 22 17673; E-mail: wim.declercq@irc.vib-UGent.be

Necrotic cell death, a controlled way of cellular explosion

Three major morphological types of cell death have been described. Type I or apoptotic cell death is mediated by caspases (a family of cysteine-dependent aspartatespecific proteases) and characterized by cellular shrinkage, membrane blebbing, chromatin condensation and DNA degradation. Type II cell death is associated with the formation of autophagic vacuoles inside the dying cell. Type III or necrotic cell death is characterized by cellular swelling, plasma membrane rupture and the subsequent loss of the intracellular content.