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The upstream and downstream regulation of the NLRP1 inflammasome in human keratinocytes

English title The upstream and downstream regulation of the NLRP1 inflammasome in human keratinocytes
Applicant Beer Hans-Dietmar
Number 197426
Funding scheme Project funding
Research institution Dermatologische Klinik Universitätsspital Zürich
Institution of higher education University of Zurich - ZH
Main discipline Experimental Cancer Research
Start/End 01.10.2020 - 30.09.2024
Approved amount 390'200.00
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All Disciplines (2)

Discipline
Experimental Cancer Research
Dermatology

Keywords (8)

inflammation; gasdermins; NLRP1; E3 ubiquitin ligase; skin cancer; UVB radiation; keratinocyte; skin

Lay Summary (German)

Lead
Die Regulation des NLRP1-Inflammasoms in menschlichen Keratinozyten
Lay summary

Karzinome der Haut stellen die häufigste Krebsart beim Menschen dar. Sie gehen aus Keratinozyten hervor, den Zellen, die die äusserste Hautschicht, auch Epidermis genannt, bilden. Die deutliche Zunahme an Hautkrebs in der Bevölkerung ist auf die gestiegene Lebenserwartung und insbesondere auf eine vermehrte Exposition der Haut mit UVB-Strahlung zurückzuführen.

Die UVB-Bestrahlung von menschlichen Keratinozyten in der Zellkultur führt zur Aktivierung des NLRP1-Inflammasoms, was wahrscheinlich den Sonnenbrand in der Haut hervorruft. Dies wird durch proinflammatorische Zytokine vermittelt, die von der Protease und Inflammasomkomponente Caspase-1 aktiviert werden. Ausserdem aktiviert Caspase-1 das Protein Gasdermin D, welches dann zum Tode der Zellen mit aktiviertem Inflammasom führt. Patienten mit aktivierenden Mutationen von NLRP1 leiden nicht nur an chronischen Entzündungen der Haut, sondern entwickeln auch mit erhöhter Wahrscheinlichkeit Karzinome.

In unserem Projekt versuchen wir einerseits, die molekularen Mechanismen der Aktivierung von NLRP1 besser zu verstehen. Andererseits werden wir die Funktionen von Gasderminen in Keratinozyten charakterisieren. Da NLRP1 nicht in der Haut der Maus vorkommt, basieren unsere Experimente auf einem in der Zellkultur gewachsenen dreidimensionalen Hautmodel, das aus direkt vom Menschen isolierten Hautzellen besteht.

Das NLRP1-Inflammasom und Gasdermine scheinen wichtige Funktionen in epidermalen Keratinozyten zu erfüllen. Die Erkenntnisse aus diesem Projekt können deshalb zur Behandlung von Entzündungen, entzündlichen Hauterkrankungen und Hautkrebs genutzt werden und somit zur Aufrechterhaltung einer gesunden Haut beitragen.


Direct link to Lay Summary Last update: 29.09.2020

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
104170 Funktion und Wirkungsmechanismus des Estrogen-responsive B box protein (EBBP) in Keratinozyten in vitro und in normaler und verwundeter Haut in vivo 01.04.2004 Project funding
117959 Roles of EBBP and its binding partner caspase-1 in the UV-induced alternative protein secretion pathway of skin-derived cells 01.10.2007 Project funding
132450 The molecular mechanisms underlying UVB-induced caspase-1-dependent apoptosis in keratinocytes 01.10.2010 Project funding
126141 Unconventional Protein Secretion (EMRC/ECORES/08-EUROMEMBRANE) 01.01.2009 Project funding (special)

Abstract

The upstream and downstream regulation of the NLRP1 inflammasome in human keratinocytesCarcinomas of the skin represent the most frequent type of skin cancer and are caused by sunlight, specifically UVB radiation. Exposure of human primary keratinocytes (HPKs) to UVB activates the NLRP1 inflammasome, a multiprotein complex able to oligomerize and recruit caspase-1, leading to the processing of inflammatory cytokines such as IL-1ß and IL-18 and the activation of cytotoxic gasdermin D (GSDMD). Mutations of the NLRP1 gene result in inflammatory skin syndromes and have been linked with the development of skin tumors. Indeed, gain-of-function mutations of NLRP1 predispose to squamous cell carcinomas (SCCs), a common type of keratinocyte-derived cancer.Although NLRP1 was the first inflammasome described in 2002, the scientific and medical interest in this protein increased just in the last few years. Still, the molecular mechanisms underlying its activation, particularly in human cells, are only partially described. It is believed that NLRP1 activation is regulated at three different stages: 1) NLRP1 undergoes proteolytic (self)processing and the resulting carboxy-terminal effector fragment is inhibited by a non-covalently interaction with the amino-terminal fragment; 2) the inhibitory amino-terminal fragment is degraded via the proteasome; 3) the remaining carboxy-terminal effector fragment induces inflammasome activation.There is evidence for a regulation of NLRP1 activity by E3 ubiquitin ligases and we will address this point by experiments based on HPKs. Moreover, we will investigate deeply the processing of NLRP1 in keratinocytes and immune cells, trying to enlighten the roles of the NLRP1 fragments and their posttranslational modifications. For this, we will modify HPK by CRISPR/Cas9, lentiviral transduction and electroporation in order to silence, overexpress and rescue NLRP1 expression, its fragments, gain-of-function and cleavage-resistant mutants and other proteins of interest.Activation of NLRP1 results in the activation of the effector protease caspase-1, which in turn activates proinflammatory cytokines and GSDMD. The latter belongs to a poorly characterized protein family consisting of five members, which are all expressed by epithelial cells and particularly keratinocytes, in a differentiation-specific manner. In immune cells, GSDMD activation induces pore formation in the outer membrane, allowing release of leaderless proteins such as IL-1ß and inducing a lytic type of cell death, termed pyroptosis. Therefore, the scientific interest in elucidating the roles of GSDMs is very high.Mice expressing a mutant form of GSDMA suffer from chronic skin inflammation, whereas GSDMB and GSDMC have been associated with (skin) cancer development. We could show that in inflammasome-activated HPKs, GSDMD is activated and supports secretion of IL-1ß, delays apoptosis and does not result in strong pyroptosis as observed in immune cells.We will analyze expression and activation of GSDMs in proliferating and differentiated HPKs as well as in normal and diseased skin, including skin cancer. Furthermore, we will characterize activation of GSDMs in UVB-irradiated HPKs and the crosstalk between the family members. Most importantly, using a three-dimensional (3D) skin model and CRISPR/Cas9-modified HPKs, we will address the differentiation-specific roles of GSDMs and aim to identify their activation mechanisms.NLRP1 and GSDM activation in keratinocytes involve pathways, which are highly relevant for inflammation, diseases and cancer development in human skin. Since these pathways are only poorly conserved in mice, HPKs in mono- or particularly in 3D culture represent ideal physiological models. This will allow to shed light on the molecular mechanisms of NLRP1 activation and on the roles of GSDMs in the skin. Our results could also give relevant hints for the involvement and characterization of these pathways in other tissues and organs.
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