ABSTRACT
The inflammasome is a complex of proteins that has a critical role in mounting an inflammatory response in reply to a harmful stimulus that compromises the homeostatic state of the tissue. The NLRP3 inflammasome, which is found in a wound-like environment, is comprised of three components: the NLRP3, the adaptor protein ASC and caspase-1. Interestingly, although ASC levels do not fluctuate, caspase-1 levels are elevated in both physiological and pathological conditions. Despite the observation that merely raising caspase-1 levels is sufficient to induce inflammation, the crucial question regarding the mechanism governing its expression is unexplored. We found that, in an inflammatory microenvironment, caspase-1 is regulated by NF-κB. Consistent with this association, the inhibition of caspase-1 activity parallels the effects on wound healing caused by the abrogation of NF-κB activation. Surprisingly, not only does inhibition of the NF-κB/caspase-1 axis disrupt the inflammatory phase of the wound-healing program, but it also impairs the stimulation of cutaneous epithelial stem cells of the proliferative phase. These data provide a mechanistic basis for the complex interplay between different phases of the wound-healing response in which the downstream signaling activity of immune cells can kindle the amplification of local stem cells to advance tissue repair.
Subject(s)
Caspase 1/physiology , Cellular Microenvironment/physiology , Dermatitis/pathology , Dermatitis/physiopathology , Keratinocytes/pathology , Animals , Caspase 8/genetics , Caspase 8/physiology , Cell Proliferation/physiology , Cells, Cultured , Disease Models, Animal , Homeostasis/physiology , In Vitro Techniques , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/physiology , Signal Transduction/physiology , Wound Healing/physiologyABSTRACT
Tissue homeostasis and regeneration are regulated by an intricate balance of seemingly competing processes-proliferation versus differentiation, and cell death versus survival. Here we demonstrate that the loss of epidermal caspase 8, an important mediator of apoptosis, recapitulates several phases of a wound healing response in the mouse. The epidermal hyperplasia in the caspase 8 null skin is the culmination of signals exchanged between epidermal keratinocytes, dermal fibroblasts and leukocytic cells. This reciprocal interaction is initiated by the paracrine signalling of interleukin 1alpha (IL1alpha), which activates both skin stem cell proliferation and cutaneous inflammation. The non-canonical secretion of IL1alpha is induced by a p38-MAPK-mediated upregulation of NALP3 (also known as NLRP3), leading to inflammasome assembly and caspase 1 activation. Notably, the increased proliferation of basal keratinocytes is counterbalanced by the growth arrest of suprabasal keratinocytes in the stratified epidermis by IL1alpha-dependent NFkappaB signalling. Altogether, our findings illustrate how the loss of caspase 8 can affect more than programmed cell death to alter the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders.
Subject(s)
Caspase 8/metabolism , Epidermis/enzymology , Wound Healing/physiology , Alstrom Syndrome , Animals , Carrier Proteins/metabolism , Caspase 1/metabolism , Caspase 8/biosynthesis , Caspase 8/genetics , Cornea/cytology , Down-Regulation , Epidermal Cells , Epidermis/metabolism , Epidermis/pathology , Inflammation/pathology , Interleukin-1alpha/metabolism , Keratinocytes/cytology , Keratinocytes/metabolism , Mesoderm/metabolism , Mice , Mice, Knockout , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Paracrine CommunicationABSTRACT
Spatial and temporal regulation of Rap1 is required for proper myosin assembly and cell adhesion during cell migration in Dictyostelium discoideum. Here, we identify a Rap1 guanosine triphosphatase-activating protein (GAP; RapGAP1) that helps mediate cell adhesion by negatively regulating Rap1 at the leading edge. Defects in spatial regulation of the cell attachment at the leading edge in rapGAP1- (null) cells or cells overexpressing RapGAP1 (RapGAP1(OE)) lead to defective chemotaxis. rapGAP1- cells have extended chemoattractant-mediated Rap1 activation kinetics and decreased MyoII assembly, whereas RapGAP1(OE) cells show reciprocal phenotypes. We see that RapGAP1 translocates to the cell cortex in response to chemoattractant stimulation and localizes to the leading edge of chemotaxing cells via an F-actin-dependent pathway. RapGAP1 localization is negatively regulated by Ctx, an F-actin bundling protein that functions during cytokinesis. Loss of Ctx leads to constitutive and uniform RapGAP1 cortical localization. We suggest that RapGAP1 functions in the spatial and temporal regulation of attachment sites through MyoII assembly via regulation of Rap1-guanosine triphosphate.
Subject(s)
Cell Adhesion , Chemotaxis , Dictyostelium/cytology , Dictyostelium/enzymology , GTPase-Activating Proteins/metabolism , Pseudopodia/enzymology , rap1 GTP-Binding Proteins/metabolism , Actins/metabolism , Animals , Cell Movement , Enzyme Activation , GTPase-Activating Proteins/chemistry , Green Fluorescent Proteins/metabolism , Kinetics , Myosin Type II/metabolism , Protein Structure, Tertiary , Protein Transport , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/enzymology , Substrate SpecificityABSTRACT
We have investigated the role of Rap1 in controlling chemotaxis and cell adhesion in Dictyostelium discoideum. Rap1 is activated rapidly in response to chemoattractant stimulation, and activated Rap1 is preferentially found at the leading edge of chemotaxing cells. Cells expressing constitutively active Rap1 are highly adhesive and exhibit strong chemotaxis defects, which are partially caused by an inability to spatially and temporally regulate myosin assembly and disassembly. We demonstrate that the kinase Phg2, a putative Rap1 effector, colocalizes with Rap1-guanosine triphosphate at the leading edge and is required in an in vitro assay for myosin II phosphorylation, which disassembles myosin II and facilitates filamentous actin-mediated leading edge protrusion. We suggest that Rap1/Phg2 plays a role in controlling leading edge myosin II disassembly while passively allowing myosin II assembly along the lateral sides and posterior of the cell.
