The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor–mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.

Macrophage JNK in Metabolic Disease: Inflammation is thought to be an important driver of diet-induced obesity and insulin resistance. Proinflammatory, M1 phenotype macrophages and the c-jun NH ₂ terminal kinases (JNK) are central players in this process. But whether JNK expression is specifically required inside macrophages is unclear. In mice containing a macrophage-specific deletion in both Jnk1 and Jnk2 , Han et al. (p. 218 , published online 6 December; see the Perspective by Ferrante Jr. ) found that the mice were protected against many of the diet-induced metabolic changes, including insulin resistance, despite similar weight gain as control mice on a high-fat diet. This protection was associated with a decrease in the presence of M1 macrophages in adipose tissue.

The notion that the cell death machinery is utilized during lens organelle degradation is supported by the observation that well characterized apoptotic substrates are cleaved during this process. Here, we test directly the role of executioner caspases (caspase-3, -6, and -7) in fiber cell differentiation. The distribution of mRNA, protein, and enzymatic activity for each caspase was determined in the mouse lens. Transcripts for all three executioner caspases were identified in lens fiber cells by real time RT-PCR, although only caspase-6 and -7 proteins were detected subsequently by Western blot analysis. Endogenous proteolytic activity was noted for caspase-3 but not caspase-6 or -7. We tested the role of executioner caspases in organelle degradation by examining lenses from mice deficient in each caspase. Knock-out lenses appeared grossly normal with the exception of caspase-3^-/- lenses, which exhibited marked cataracts at the anterior lens pole. The distribution of lens organelles was mapped by confocal microscopy. There was no significant difference in the size of the lens organelle-free zone (OFZ)1 between wild-type and knock-out lenses. In response to treatment with staurosporine, caspase-3 and -6 (but not caspase-7) enzymatic activities were induced. We generated double knock-out animals to examine the phenotype of lenses deficient in both caspase-3 and -6. Histological examination of such lenses indicated the presence of a properly formed OFZ. Thus, no single executioner caspase (nor a combination of caspase-3 and -6) is required for organelle loss, although caspase-3 activity may be required for other aspects of lens transparency.

P42mapk [mitogen activated protein (MAP) kinase; extracellular signal-regulated protein kinase (ERK)] is a serine/threonine-specific protein kinase that is activated by dual tyrosine and threonine phosphorylation in response to diverse agonists. Both the tyrosine and threonine phosphorylations are necessary for full enzymic activity. A MAP kinase activator recently purified and cloned has been shown to be a protein kinase (MAP kinase kinase) that is able to induce the dual phosphorylation of MAP kinase on both the regulatory tyrosine and threonine sites in vitro. In the present paper we have utilized MAP kinase mutants altered in the sites of regulatory phosphorylation to show, both in vivo and in vitro, that phosphorylation of the tyrosine and the threonine can occur independently of one another, with no required order of phosphorylation. We also utilized kinase-defective variants of MAP kinase with mutations in either the ATP-binding loop or the catalytic loop, and obtained data suggesting that the activity or structure of the catalytic loop of MAP kinase plays an important role in its own dual phosphorylation.

Members of the caspase family are essential for many apoptotic programs. We studied mouse embryonic fibroblasts (MEFs) deficient in caspases 3 and 7 and in caspase 9 to determine the role of these proteases in endoplasmic reticulum (ER) stress-induced apoptosis. Both caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs were resistant to cytotoxicity induced via ER stress and failed to exhibit apoptotic morphology. Specifically, apoptosis induced by increased intracellular calcium was shown to depend only on caspases 3 and 9, whereas apoptosis induced by disruption of ER function depended additionally on caspase 7. Caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs also exhibited decreased loss of mitochondrial membrane potential, which correlated with altered caspase 9 processing, increased induction of procaspase 11, and decreased processing of caspase 12 in caspase 3(-/-)/caspase 7(-/-) cells. Furthermore, disruption of ER function was sufficient to induce accumulation of cleaved caspase 3 and 7 in a heavy membrane compartment, suggesting a potential mechanism for caspase 12 processing and its role as an amplifier in the death pathway. Caspase 8(-/-) MEFs were not resistant to ER stress-induced cytotoxicity, and processing of caspase 8 was not observed upon induction of ER stress. This study thus demonstrates a requirement for caspases 3 and 9 and a key role for the intrinsic pathway in ER stress-induced apoptosis.