Inactivation of the Cytoprotective Major Vault Protein by Caspase-1 and -9 in Epithelial Cells during Apoptosis.

Inflammasome activation induces caspase-1-dependent secretion of the proinflammatory cytokine IL-1ß. In addition, caspase-1 activates the protein GSDMD in immune cells, causing pyroptosis, a lytic type of cell death. In contrast, UVB irradiation of human primary keratinocytes induces NLRP1 inflammasome activation, cytokine secretion, and caspase-1-dependent apoptosis, rather than pyroptosis. Here, we addressed the molecular mechanisms underlying the role of caspase-1 in UVB-induced cell death of human primary keratinocytes. We show that GSDMD is a poor substrate of caspase-1 in human primary keratinocytes and that its activation upon UVB irradiation supports secretion of IL-1ß. We screened for novel substrates of caspase-1 by a mass spectrometry-based approach and identified the specific cleavage of the major vault protein (MVP) at D441 by caspase-1 and -9. MVP is the main component of vaults, highly conserved ribonucleoprotein particles, whose functions are poorly understood. Cleavage of MVP is a common event occurring in human primary keratinocytes and fibroblasts undergoing apoptosis induced by different stimuli. In contrast, MVP cleavage could not be detected in pyroptotic cells. Cleavage of MVP by caspase-1 and -9 inactivates this cytoprotective protein. These results demonstrate a proapoptotic activity of caspase-1 and a crosstalk with caspase-9 upon inactivation of the cytoprotective MVP in apoptotic epithelial cells.

The NLRP1 Inflammasome Pathway Is Silenced in Cutaneous Squamous Cell Carcinoma.

The inflammasome protein NLRP1 is an important innate immune sensor in human keratinocytes, and, together with ASC and caspase-1, it mediates the activation and secretion of the proinflammatory cytokines IL-1ß and IL-18. These cytokines and inflammasomes can have partly opposing roles during tumorigenesis in mice. In contrast, ASC expression is impaired in different types of cancer in humans. In this study, we analyzed inflammasome activation and expression of inflammasome proteins, including their downstream cytokines, in squamous cell carcinomas, a type of nonmelanoma skin cancer derived from keratinocytes. We assessed mRNA and protein levels in human primary keratinocytes and skin carcinoma-derived SCC cell lines and detected a strong down-regulation of expression of NLRP1 inflammasome components, as well as reduced expression of the proinflammatory cytokines proIL-1ß and proIL-1α. Protein levels of NLRP1, ASC, caspase-1, and proIL-1ß were reduced in patient-derived SCC biopsy samples compared with healthy skin. Furthermore, the results suggest that expression of PYCARD (ASC), CASP1, IL1B, and NLRP1 is silenced by methylation in SCC cell lines. In conclusion, the down-regulation of the inflammasome pathway in SCCs might favor late tumor development in human skin.

Genome Editing of Human Primary Keratinocytes by CRISPR/Cas9 Reveals an Essential Role of the NLRP1 Inflammasome in UVB Sensing.

By forming a protective barrier, epidermal keratinocytes represent the first line of defense against environmental insults. UVB radiation of the sun is a major challenge for the skin and can induce inflammation, aging, and eventually skin cancer. UVB induces an immune response in human keratinocytes resulting in activation and secretion of the proinflammatory cytokines proIL-1ß and -18. This is mediated by an assembly of protein complexes, termed inflammasomes. However, the mechanisms underlying sensing of UVB by keratinocytes, and particularly the types of inflammasomes required for cytokine secretion, are a matter of debate. To address these questions, we established a protocol that allows the generation of CRISPR/Cas9-targeted human primary keratinocytes. Our experiments showed an essential role of the NLRP1 rather than the NLRP3 inflammasome in UVB sensing and subsequent IL-1ß and -18 secretion by keratinocytes. Moreover, NLRP1 but not NLRP3 was required for inflammasome activation in response to nigericin, a potassium ionophore and well-established NLRP3 activator in immune cells. Because the CRISPR/Cas9-targeted cells retained their full differentiation capacity, genome editing of human primary keratinocytes might be useful for numerous research and medical applications.

Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome.

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor.

Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1ß. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

The Arabidopsis STRESS RESPONSE SUPPRESSOR DEAD-box RNA helicases are nucleolar- and chromocenter-localized proteins that undergo stress-mediated relocalization and are involved in epigenetic gene silencing.

DEAD-box RNA helicases are involved in many aspects of RNA metabolism and in diverse biological processes in plants. Arabidopsis thaliana mutants of two DEAD-box RNA helicases, STRESS RESPONSE SUPPRESSOR1 (STRS1) and STRS2 were previously shown to exhibit tolerance to abiotic stresses and up-regulated stress-responsive gene expression. Here, we show that Arabidopsis STRS-overexpressing lines displayed a less tolerant phenotype and reduced expression of stress-induced genes confirming the STRSs as attenuators of Arabidopsis stress responses. GFP-STRS fusion proteins exhibited localization to the nucleolus, nucleoplasm and chromocenters and exhibited relocalization in response to abscisic acid (ABA) treatment and various stresses. This relocalization was reversed when stress treatments were removed. The STRS proteins displayed mis-localization in specific gene-silencing mutants and exhibited RNA-dependent ATPase and RNA-unwinding activities. In particular, STRS2 showed mis-localization in three out of four mutants of the RNA-directed DNA methylation (RdDM) pathway while STRS1 was mis-localized in the hd2c mutant that is defective in histone deacetylase activity. Furthermore, heterochromatic RdDM target loci displayed reduced DNA methylation and increased expression in the strs mutants. Taken together, our findings suggest that the STRS proteins are involved in epigenetic silencing of gene expression to bring about suppression of the Arabidopsis stress response.

Modulation of Rack-1/PKCßII signalling by soluble AßPPα in SH-SY5Y cells.

The soluble amyloid ß precursor protein α (sAßPPα) released after α-secretase cleavage of the amyloid ß precursor protein (AßPP) has several functions including modulation of neuronal excitability and synaptic plasticity; it has been suggested that some of these effects are mediated by activation of NF-κB via induction of PI3K/Akt signaling pathway. We have recently described the presence of several consensus binding sites of c-Rel transcription factor in the promoter region of the GNB2L1 gene, coding for the Receptor for Activated C Kinase -1 (RACK-1). We investigated whether sAßPPα could influence the expression of RACK-1 through NF-κB involvement. Our data demonstrate that sAßPPα regulates RACK-1 gene expression through PI3K/Akt-dependent pathway, inducing c-Rel nuclear translocation and NF-κB activation. Since RACK-1 is the scaffold of protein kinase C ßII (PKCßII), we turned our attention to this kinase in order to evaluate whether sAßPPα could also influence PKCßII signalling demonstrating that sAßPPα induces PKCßII translocation and interaction with its scaffold with consequent RACK-1/PKCßII complex increase in membrane. Altogether these results suggest the existence of an interesting loop between the functions of the metabolic products of AßPP and the role of PKC and that the impact of a dysregulated AßPP metabolism occurring in several conditions (from physiological aging to injury response) may have consequences on the potential protective functions of the non amyloidogenic sAßPPα.