The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.

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The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response. Executioner caspases mediate cell death during apoptosis (45). Of these, caspase-3 has the ability to cleave the majority of the caspase substrates (43), and its activity is required for the induction of cell death in response to many apoptotic stimuli (1). While executioner caspases are indispensable for apoptosis, there are situations when their activation does not lead to death. For example, healthy dividing cells can weakly activate caspase-3 in response to mild stresses (47). Caspase-3 also participates, in an apoptosisindependent manner, in T and B cell homeostasis (35,46), in microglia activation (6), in long-term depression (26), and in muscle (17), monocyte (44), embryonic stem cell (18), and erythroid cell (13) differentiation. However, it remains unclear how activation of caspase-3 under these conditions does not eventually lead to cell death (1, 24). Cells could have an intrinsic ability to tolerate low caspase activity by constitutively expressing antiapoptotic molecules, such as members of the inhibitors of the apoptosis protein family, or may stimulate antiapoptotic pathways in parallel to caspase activation (24). Alternatively, the caspases themselves might activate prosurvival pathways, in particular, when they are mildly stimulated. Indeed, there is evidence in cultured cells that caspase-3 mediates neuroprotection after preconditioning (30) and that caspase-3 activity turns on the antiapoptotic Akt kinase following partial cleavage of the RasGAP protein (47). Other caspase substrates that could potentially induce protective signals once cleaved include p27 kip1 (14), Lyn (28), synphilin-1 (19), and Rb (42), yet the physiological importance of these cleaved substrates has not been evaluated to date.In the present study, we have investigated the role played by caspase-3 and its substrate p120 RasGAP in the induction of the antiapoptotic Akt kinase in stressed tissues in vivo. MATERIALS AND METHODS Caspase-3-KO mice. B6.129S1...

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RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apoptosis. Therefore, the role of the second caspase-mediated cleavage of RasGAP is to allow the inactivation of the antiapoptotic function of fragment N so that caspases are no longer hampered in their ability to kill cells.