Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731–6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8–induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.
SummaryIterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.
SUMMARY The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3, or the profission gene drp-1, are not required for apoptosis activation in C. elegans. However minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9, and downstream of the CED-3 caspase, to promote elimination of mitochondria in dying cells, an event that could facilitate cell death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1’s proapoptotic function but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell death execution downstream of caspase activation.
BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.Programmed cell death is characterized by a series of morphological and structural changes culminating in the coordinated packaging of cellular contents into apoptotic bodies, which are ultimately eliminated via phagocytosis by neighboring cells. Early events in this process typically include cell rounding, loss of phospholipid asymmetry in the cell membrane, extensive cytoplasmic membrane blebbing and fragmentation, nuclear pyknosis, and internucleosomal DNA cleavage (26). Although much remains to be learned about the mechanisms underlying these events, programmed cell death is achieved in most cell death pathways as a consequence of the proteolytic cleavage of a diverse array of structural and regulatory proteins by the executors of apoptosis, the caspase family of cysteine proteases (33,46,48). Several of the caspase targets are known to have critical roles in at least some of the apoptotic processes. These targets include the DFF40/CAD inhibitor, DFF45/ICAD, which plays a role in the fragmentation of DNA (23, 36), and the actin-associated capping protein gelsolin, which plays a role in cytoplasmic membrane blebbing (17). In addition, the activation of several kinases by caspase cleavage, including PAK2 (20, 34) and the Ste20-related kinases MST1 (14,1...
BAP31 is an integral protein of the endoplasmic reticulum membrane and a substrate of caspase-8. Here, we describe the procaspase-8 isoform, procaspase-8L, which is ubiquitously expressed and selectively recruited to the BAP31 complex in response to apoptotic signaling by E1A. Procaspase-8L is characterized by the N-terminal extension (Nex) domain, which extends procaspase-8͞a at the N terminus and is required for selective association of procaspase-8L with the BAP31 complex. Gene deletion identified BAP31 and related BAP29 as required for processing of procaspase-8L in response to E1A, by a FADD-independent mechanism that was blocked by BCL-2. Further, Bap29,31 deletion, as well as a Nex-domain dominant-negative mutant, curtailed the activation of downstream caspases (IETDase and DEVDase) and cell death in response to E1A. Preferential recruitment of procaspase-8L by the BAP31 complex at the endoplasmic reticulum suggests an additional pathway for regulating initiator caspase-8 during apoptosis.
Dual activity of ACC inhibitors provides anti-fibrotic benefit Hepatocytes Hepatic stellate cells ↓ Lipotoxicity ACC inhibitor ↓ Lipogenesis ↑ Fatty acid oxidation ↓ Glycolysis, mitochondrial respiration ↓ Collagen production ↓ α-SMA expression ↓ HSC activation Highlights ACC inhibition blocks TGF-b-induced hepatic stellate cell activation. ACC inhibition blocks tension-mediated activation of primary rat and human hepatic stellate cells. Inhibiting de novo lipogenesis targets hepatic stellate cells' reliance on increased glycolysis and oxidative phosphorylation. ACC inhibition in vivo significantly reduces fibrosis in 4 models of non-alcoholic steatohepatitis.
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