HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

Apoptosis and non-inflammatory phagocytosis can be induced by mitochondrial damage without caspases.

A central issue regarding vertebrate apoptosis is whether caspase activity is essential, particularly for its crucial biological outcome: non-inflammatory clearance of the dying cell. Caspase-9 is required for the proteolytic cascade unleashed by the mitochondrial outer membrane permeabilization (MOMP) regulated by the Bcl-2 protein family. However, despite the severely blunted apoptosis in cells from Casp9(-/-) mice, some organs with copious apoptosis, such as the thymus, appear unaffected. To address this paradox, we investigated how caspase-9 loss affects apoptosis and clearance of mouse fibroblasts and thymocytes. Although Casp9(-/-) cells were initially refractory to apoptotic insults, they eventually succumbed to slower caspase-independent cell death. Furthermore, in gamma-irradiated mice, the dying Casp9(-/-) thymocytes were efficiently cleared, without apparent inflammation. Notably, MOMP proceeded normally, and the impaired mitochondrial function, revealed by diminished mitochondrial membrane potential (DeltaPsi(m)), committed cells to die, as judged by loss of clonogenicity. Upon the eventual full collapse of DeltaPsi(m), presumably reflecting failure of respiration, intact dying Casp9(-/-) cells unexpectedly exposed the prototypic 'eat-me' signal phosphatidylserine, which allowed their recognition and engulfment by phagocytes without overt inflammation. Hence, caspase-9-induced proteolysis accelerates apoptosis, but impaired mitochondrial integrity apparently triggers a default caspase-independent program of cell death and non-inflammatory clearance. Thus, caspases appear dispensable for some essential biological functions of apoptosis.

Peroxisome proliferator-activated receptor gamma ligands differentially modulate muscle cell differentiation and MyoD gene expression via peroxisome proliferator-activated receptor gamma -dependent and -independent pathways.

The effects of distinct classes of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPARgamma ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and alpha-actin. In contrast, two synthetic PPARgamma ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type- and promoter-specific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPARgamma on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPARgamma. Interestingly, a transdominant inhibitor of PPARgamma (hPPARgamma2Delta500) had no effect on the 15d-PGJ2-dependent repression of MyoDLuc expression but overcame L-805645/PPARgamma-dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPARgamma ligands may repress MyoD gene expression through PPARgamma-dependent and -independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPARgamma.