Introduction

For all multicellular organisms, cell number control is essential for proper organ formation during development, and for cellular homeostatsis in adults.¹ Such a critical task requires a delicate balancing act of cell proliferation and, as realized more recently, cell death. While much attention in the past has been given to the molecular mechanisms that control how cells divide and propagate, recent efforts unveiled an equally elaborate cellular machinery that governs the process of cell suicide, also known as apoptosis. At the very center of this tightly-regulated process is a group of intracellular cysteine proteases called caspases for cysteinyl aspartate-specific proteinases.² Upon receiving apoptotic stimuli, these otherwise latent proteases become activated and carry out cell destruction through proteolytically cleaving specific intracellular targets.

To date, approximately a dozen mammalian caspases have been identified through both genetic and biochemical means.³ The presence of multiple caspases therefore raised the critical question of how individual caspases contribute to apoptosis in vivo. To definitely address this issue, we and others have generated several lines of caspase-deficient mice using gene targeting strategy.⁴ Analyses of these caspase-deficient mice have contributed significantly to our current understanding of how caspases function in vivo. For example, studies using caspase-3^−/− cells unequivocally established caspase-3 as the key caspase that mediates the execution of apoptosis in mammals.⁵ Similarly, generation of caspase-9-deficient mice confirmed the existence of ‘caspase cascade’ regarding caspase activation during apoptosis, as suggested by elegant in vitro studies.^6,7 In this chapter, our aim is to provide an overview of what we have learned from caspase knockout mice, with a focus on some of the more surprising findings revealed.

Overview of Caspase-Deficient Mice

Gene targeting in the past decade has become the standard approach to examine gene function in physiological settings. Among the ten known murine caspases, all but caspase-14 have been knocked out (see Table 1). Given the confusing nature of caspase nomenclature due to historical reasons, it is worth pointing out that caspase-11 and -12 have only been identified in mouse and are most likely the murine homologues of human caspase-4 and -5, respectively. In addition, caspase-10 probably only exists in human, and recent reports have argued that caspase-13 is actually a bovine caspase gene, rather than a new human caspase, as originally reported.⁸

Table 1

Murine caspases and caspase-deficient mice.

Based on sequence homology, prodomain function and substrate specificity, caspases are generally categorized into three groups: initiator caspases (caspase-8, -9 and -2), effector caspases (caspase-3, -6 and -7) and caspases that are primarily involved in mediating inflammatory responses (caspase-1, -11 and -12).⁹ According to the proposed ‘cascade model’ of caspase activation, multiple caspases are activated in the following stepwise manner during apoptosis.² In response to a given apoptotic stimulus, one upstream initiator caspase is first activated through adaptor-mediated autoprocessing. The activated initiator caspase in turn proteolytically activates several downstream effector caspases, whose activation leads to the final destruction of apoptotic cells by cleaving various cellular targets. One can therefore predict that deficiency in individual caspases will likely result in defect in the initiation or execution of apoptosis, or in inflammatory responses.

The generation of mice deficient in these caspases has by and large confirmed such a prediction with a few exceptions. For example, deficiency in the initiator caspases-8 or -9 results in nearly complete block of downstream caspase activation and apoptosis induced through their respective pathways, demonstrating that initiator caspases are absolutely required for the progression of caspase-mediated cell death.^6,10 However, only caspase-3,¹¹ but not caspases-6 or -7, appears to be required for apoptosis execution (unpublished data), raising the possibility of functional redundancy among downstream effort phase caspases or that caspase-6 and -7 are involved in other cellular functions. With respect to caspases that are thought to mediate inflammation, caspase-1^−/− and -11^−/− mice both indeed exhibit defective inflammatory response due to their inability to process and secret proinflammatory cytokine interleukin-1β and -1α.^12–14 Caspase-12 deficiency, on the other hand, had no apparent effect on inflammation, but instead resulted in defect in apoptosis initiated through the endoplasmic reticulum (ER) pathway.¹⁵

Caspase Deficiency and Mammalian Development

Developmentally regulated apoptosis plays a prominent role during mammalian embryogenesis.¹ Complex tissue/organ formation often involves generation of excessive cells and their subsequence removal by apoptosis to ensure fine-tuning of the process. Thanks to the generation of various caspase-deficient mice, it is now clear that several caspases are essential for mammalian development.

The best-studied example is the involvement of caspase-9 and -3 pathway in neuronal development. Mice deficient in either caspase-3 or 9 exhibit similar developmental blockage characterized by perinatal lethality.^5–7 Histological analysis of the surviving newborns revealed various neural phenotypes that likely resulted from supernumerary neurons during CNS development. For example, contrary to the smooth surface of the cerebrum mantel in normal rodents, the caspase-3^−/− mouse brain exhibited multiple indentations reminiscent of gyria structures found in higher vertebrates, indicative of excessive brain cell mass. Also, BrdU-negative ectopic cell populations were frequently observed in various areas around the hippocampus region, some of which were even capable of adopting proper cellular deployment such as the formation of a ‘double cortex’. Further examination of developing embryos deficient in either caspase-3 or -9 indeed confirmed our hypothesis that deletion in either caspase-3 or -9 resulted in severe developmental block due to aberrant neuronal apoptosis. As early as E12, a pronounced increase in cellularity was already evident in the proliferative zone along the ventricular area of the mutant embryos. Toluidine blue staining revealed that while pyknotic clusters could routinely be seen at the interventricular junctions in the wild-type E12 embryos, apoptotic cells were absent in the same areas of the caspase-3^−/− or -9^−/− embryos. Thus, the increased cellularity in the CNS associated with caspase-3 or -9 deficiency resulted directly from lack of apoptosis during early neuronal development. The presence of these superfluous cells was so profound that blockage of certain brain structures such as the aqueduct was frequently observed at later developmental stage.

In addition to caspase-3 and -9, knockout studies have also identified a few other caspases that are required for proper mammalian development, although the underlying mechanisms of their involvement are poorly understood. For example, deletion of caspase-7 resulted in very early developmental arrest during embryogenesis, yet the precise nature and cause of such arrest remains unknown (unpublished data). Also, mice deficient in caspase-8, the initiator caspase of the death receptor apoptotic pathway, are embryonically lethal with impaired cardiac development and abnormal erythrogenesis.¹⁰ It is not clear, however, whether these defects are a primary or secondary effect resulting from caspase-8 deficiency and whether they reflect defective apoptosis during development. In fact, null mutation of FLIP,¹⁶ the ‘decoy’ caspase-8 like molecule that antagonizes caspase-8 function in the death receptor apoptotic pathway, resulted in nearly identical developmental phenotypes,¹⁷ therefore arguing for a critical role of the FADD/caspase-8/FLIP pathway during mammalian development that is not related to apoptosis.

The generation of these caspase knockout mice has not only provided critical insights into the role of caspases in mammalian development, but also revealed a few unexpected findings. First, genetic background strongly influences phenotypic penetrance of caspase-3-deficient mice.¹⁸ As originally reported, caspase-3^−/− mice under the mixed 129 and C57BL/6 background exhibited perinatal lethality and varied severity in their neuronal phenotype among individual animals. When backcrossed onto the C57BL/6 background, however, caspase-3 knockout mice can survive through adulthood with no obvious CNS defect. On the other hand, caspase-3^−/− mice bred onto the pure 129 background are completely embryonic lethal, similar to that seen in the caspase-9-deficient animal (Kevin Roth, personal communication). Since the greatly improved survivability of caspase-3 KO under C57BL/6 was not observed with caspase-9^−/− mice, it is likely that a genetic modifier capable of modulating the caspase-9/-3 activation pathway in developing neurons functions downstream of caspase-9, but either upstream or at the same level of caspase-3.

The second surprise is the apparent tissue-specificity of the developmental phenotypes observed in these caspase-deficient mice, given the overlapping expression pattern, at least in adult animals, of these caspases in most, if not all tissues. Although one might argue for possible spatial and temporal regulation of expression among individual caspases during embryogenesis, an alternate explanation, perhaps more intriguing, is the possibility that distinct caspase pathways are being activated in different tissues/organs in response to certain yet to be discovered developmental cues.

Caspases in Apoptosis Execution

Apoptotic cells are characterized by a number of distinct morphological and biochemical changes such as cell shrinkage, cytoplasmic bleb formation, nuclear condensation and DNA fragmentation.¹⁹ Previous studies using the broad-spectrum caspase inhibitor zVAD-fmk have concluded that most, if not all, of these cellular alterations can be attributed to caspase activity.²⁰ Such studies, however, have largely failed to distinguish the individual contribution of each effector caspase. Only with the generation of individual caspase knockout strains have we begun to address the precise involvement of each caspase during apoptosis execution.

So far, caspase-3 has clearly emerged as the single most important caspase during the execution phase of apoptosis. Using cells derived from caspase-3-deficient mice, we and others have shown that dying caspase-3^−/− cells undergo an aberrant form of apoptosis, exhibiting drastically delayed cellular changes such as cytoplasmic bleb formation, nuclear and DNA fragmentation.^11,21 We further demonstrated that caspase-dependent cleavages of several intracellular proteins such as fodrin-α, DFF45/ICAD, lamin B and gelsolin, whose degradations have been linked to the various morphological and biochemical features associated with apoptosis, were impaired in caspase-3^/ thymocytes undergoing apoptosis.¹¹ These results strongly suggest that caspase-3 is the major effector caspase responsible for many, but not all, of the proteolytic events leading to cellular destruction. Importantly, similar results were also obtained in the human breast carcinoma line MCF-7,²² which is ‘naturally’ deficient in caspase-3 expression, not only validating the results obtained with knockout cells, but also indicating that the essential role of caspase-3 in apoptosis execution is evolutionarily conserved.

In addition to caspase-3, caspase-6 and -7 are also thought to be important effector caspases given their sequence similarity to caspase-3.²³ In vitro experiments has previously suggested that caspase-6 was critical for the proteolysis of nuclear structural proteins such as lamin A and NUMA.²⁴ Thus, it came as quite a surprise when caspase-6-deficient mice exhibited no defect in apoptosis and dying caspase-6^−/− cells underwent normal nuclear breakdown (unpublished data). Furthermore, cleavages of a number of caspase substrates in apoptotic caspase-6^−/− thymocytes, including lamin B, also appeared normal, indicating that caspase-6 is dispensable for apoptosis execution. As for caspase-7, while the early embryonic lethality of caspase-7 KO mice has significantly hindered the scope of their characterization, the generation of caspase-7^−/− embryonic stem (ES) cells using G418 selection has allowed us to examine its involvement during apoptosis execution. To our surprise, deletion of caspase-7 in ES cells did not result in any obvious defect in cell death or substrate cleavage induced by various stimuli such as UV irradiation and etopside, suggesting that caspase-7 is not required for apoptosis execution, at least in ES cells.

Taken together, gene targeting of caspase-3, -6 and -7 has provided critical insight into the role (and also lack of role) of these caspases in apoptosis execution. It confirmed previous speculation that caspase-3 is the most important contributor of caspase-mediated proteolysis of cellular targets during apoptosis. At the same time, however, it also raised a number of questions as to what other caspases are involved in the effector phase and what role, if any, caspase-6 and -7 have. Since it is clear that caspase-3 is not required for the cleavage of all substrates such as PARP-1, previously thought a caspase-3 target,^25,26 which caspase(s), then, is responsible for their proteolysis? Although one can argue for compensation by caspase-6 or -7 in the absence of caspase-3, we do not favor this possibility since the cleavage of many substrates in caspase-3^−/− cells are defective. It is an unlikely scenario that selective compensation by other caspases on the cleavage of certain substrates, but not others, would occur.

The apparent lack of function for caspase-7 during apoptosis execution seen in caspase-7^−/− ES cells is perhaps most surprising. All in vitro studies have concluded that the enzymatic activity of caspase-7 was indistinguishable from that of caspase-3.²⁷ Furthermore, unlike caspase-6 whose activation requires caspase-3 activity, caspase-7 is usually activated concurrently with caspase-3 in wild-type cells and caspase-7 activation appears normal in apoptotic caspase-3^−/− cells (unpublished data). Based on these results, one would predict that caspase-7 should be able to compensate for caspase-3 in substrate cleavage in apoptotic caspase-3 null cells. It is therefore perplexing why caspase-7 failed compensate for caspase-3 deficiency in substrate cleavage. Our current hypothesis is that perhaps caspase-3 and -7 are differentially compartmentalized within the cell and the defective substrate cleavage observed in caspase-3^−/− cells despite caspase-7 activation is due to inaccessibility of these substrates to caspase-7. The validity of this hypothesis should be testable experimentally.

The Plasticity of Caspase Activation

Much effort in the past few years has gone into understanding how caspases are activated during apoptosis. Results from both in vitro and in vivo studies support a ‘branched cascade model’ of caspase activation.^28,29 Briefly, apoptotic signaling first triggers adaptor-mediated direct activation of its corresponding initiator caspase such as caspase-8 or -9, likely through an autoprocessing mechanism. Activated initiator caspase in turn proteolytically activates two downstream effector caspases including caspase-3 and -7. Interestingly, the activation of the other presumed effector caspase, caspase-6, requires caspase-3 activity. To date, two major caspase-activating pathways have been characterized in detail, namely the extrinsic pathway of caspase activation induced by death receptors and the intrinsic pathway of caspase activation involving mitochondria participation.

More recent evidence, however, suggested that the cellular pathways of caspase activation are more flexible than originally thought. One good example is the caspase activation triggered by death receptor signalling.³⁰ While death receptors universally induce FADD-dependent recruitment and activation of the initiator caspase-8 upon ligand engagement, the subsequent events could diverge depending on the cellular context. In addition to directly cleaving and activating caspase-3, caspase-8 can also trigger the intrinsic pathway through proteolytically activating a pro-apoptotic member of the Bcl2 family, Bid, whose activation and subsequent translocation into the mitochondria result in cytochrome c release and caspase-9 activation.^31,32 The relevant contribution of the mitochondrial pathway to death receptor signaling is apparently cell type specific, but the underlying molecular basis remains poorly understood.³³

To investigate potential plasticity of caspase activation in vivo, we took advantage of a well-established in vivo model of Fas-induced hepatocyte apoptosis.³⁴ Previous studies have demonstrated that injection of the agonistic anti-Fas antibody Jo-2 induced massive hepatocyte apoptosis and led to rapid animal death. It has also been shown that Jo-2 induced liver damage, and lethality is dependent on Bid-mediated mitochondrial pathway of caspase activation.³⁵ Indeed, Jo-2 injection into wild-type mice resulted in Bid translocation-induced cytochrome c release in hepatocytes and subsequent activation of both caspase-9 and -3. The Jo-2-induced caspase activation pattern in caspase knockout mice, however, altered dramatically and demonstrated a great deal of flexibility.³⁶ Deficiency in caspase-3, for example, resulted in compensatory activation of caspase-7 and -6 following Bid-induced caspase-9 activation. If the compensation of caspase-3 by caspase-7 and -6 is perhaps somewhat expected, the caspase activation pattern elicited in Jo-2-treated caspase-9 knockout mice offered a total surprise. In the absence of caspase-9, Bid translocation-induced mitochondrial events triggered significant activation of caspase-2 and -6, revealing an alternative caspase-activating pathway that was previously unknown. Overall, these results strongly suggest that caspase activation in vivo is not a rigid process, but rather consists of multiple pathways capable of compensating one another.

In addition to the compensatory mechanisms revealed by caspase-3 and -9 knockout mice, the early lethality in caspase-7 KO also suggests a novel pathway of caspase activation. Since no other caspase knockout exhibits developmental block at a similar stage, it is unlikely that one of the known initiator caspases is involved in the developmental step that requires caspase-7. Thus, caspase-7 may function as both an initiator and an effector caspase in response to the developmental cue and undergo direct activation.

Caspase Beyond Death

Caspase function has clearly expanded during evolution. While ced-3, the likely only functioning caspase in C. elegans, has no other apparent function other than to mediate programmed cell death during nematode development,³⁷ both in vitro and in vivo studies suggested that not all mammalian caspases function in apoptosis. In fact, caspase-1 was first identified as the interleukin-1β converting enzyme (ICE) and knockout studies have confirmed its main function as a critical mediator of inflammatory responses.^12,13 An almost identical role for caspase-11 has also been established based on the characterization of caspase-11 knockout mice, although the precise functional relationship between caspase-1 and -11 in IL-1 production has yet to be worked out.¹⁴

Additional involvement of caspases in biological processes other than apoptosis and inflammation has been further implicated from studies on caspase knockout mice.⁴ As mentioned previously, null mutation of caspase-8 results in embryonic lethality characterized by defective cardiac development, and several lines of evidence suggest that the developmental phenotype seen in caspase-8^−/− embryos was probably not due to defective apoptosis. First, apoptosis is not known to play a prominent role during cardiac development. More importantly, FLIP deficiency, which resulted in enhanced apoptosis through death receptors, exhibited essentially the same developmental defects,¹⁷ strongly suggesting the myocardiac abnormality as a result of caspase-8 is not apoptosis related. Indeed, a number of in vitro studies have argued that caspase-8-mediated FLIP proteolysis could activate the NF-κB pathway and was required for T cell proliferation.^38,39

Another clue for alternative caspase function comes from analysis of caspase-6-deficient mice. While no apparent apoptosis defect has been identified in these mutant mice, caspase-6^−/− B cells appear somewhat abnormal phenotypically with no or very low expression of surface CD23 (unpublished data). Consistent with the role of caspase-6 in CD23 expression in B cells, CD40L-induced CD23 expression in WEHI 231 immature B cell line could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk, suggesting a potential role for caspase activity in B cell maturation. The functional significance of defective CD23 expression in caspase-6^−/− B cells is currently under investigation.

Future Perspectives

The cloning of multiple mammalian caspases has presented us with the critical question of what in vivo role individual caspases plays and how these caspases functionally relate to each other in biological processes. Thanks to the generation of various caspase knockout mice, significant strides have been made toward understanding the contribution of many caspases during mammalian development, apoptosis and inflammatory responses. Despite the progress, we are still faced with a number of important questions revealed by these knockout mice regarding the function of certain caspases and how caspase activities are regulated in vivo.

First, the early embryonic lethality associated with deficiency in caspase-8, -9 or -7 has greatly limited the characterization of these caspase knockout mice. As a result, much remains unknown about the function of caspase-8, -9 and -7. For example, is caspase-8 required for T cell development and function, as one might expect from in vitro studies suggesting that caspase-8 activity is critical for T cell proliferation?³⁸ Similarly, despite our knowledge of caspase-9's involvement in neuronal apoptosis during development, very little is known about whether caspase-9 activity is absolutely required for apoptosis induced by various stimuli in other cell types. Obviously, the answer to such questions calls for the generation of conditional knockout mice, which would also allow assessment of the involvement of caspase-8 and -9 in various mouse models of human diseases. As the technology for creating both tissue-specific and temporally regulated conditional knockout mice continues to improve, we should expect a great deal of critical insights into the function of these caspases in physiological and pathological settings.

One intriguing finding from caspase knockout mice is the strong influence strain background exerts on the developmental phenotypes observed in caspase-3-deficient mice. As discussed before, while caspase-3 deficiency in the C57BL/6 background leads to dramatically improved animal survival compared to the original reported 129 and C57BL/6 mixed background, all pure 129 background caspase-3^−/− mice are embryonically lethal. Interestingly, the requirement of caspase-3 for mediating the various morphological and biochemical changes associated with apoptosis did not alter with different backgrounds, suggesting that the genetic modifier is likely influencing the regulation of neuronal apoptosis during development, rather than exerting a direct effect on the caspase activation pathway. In past few years, phenotypic variation of gene knockout mice due to genetic background has been increasingly noticed and several attributing genetic modifiers have been isolated in a few cases through extensive backcrossing and phenotypic analysis.^40,41 The continuously improved sequence coverage of the entire mouse genome should greatly facilitate the isolation of genetic modifier that determines the viability of caspase-3-deficient mice.

Finally, despite our lack of understanding of its precise molecular mechanism, the compensatory pathways of caspase activation observed in caspase-3 and -9 knockout mice will likely have important implications.³⁶ As caspases have become attractive targets for therapeutic interventions for various diseases in which excessive apoptosis has been attributed to pathogenesis, selective inhibition of one or two upstream caspases that are involved in a particular disease has been suggested to be the ideal strategy. Our discovery, however, clearly indicates that cells have the capacity to activate caspases through compensatory pathways and efficient inhibition of caspase activity would require blocking such alternative pathways as well. On the other hand, mechanistic insights into how compensatory activation of caspase-2 and -6 can be achieved in the absence of caspase-3 and -9 may also provide the new means to ‘jumpstart the death engines’ in apoptosis-resistant tumor cells, whose regular apoptotic machinery, including caspases activating pathway, is almost certainly perturbed.

Acknowledgments

All the unpublished studies mentioned in this article were carried out in Dr. Richard Flavell's laboratory. I am deeply indebted to the insightful guidance and wonderful opportunity Dr. Flavell has provided to me over the course of my study under his supervision. I would also like to thank various members of laboratory and outside collabortors for their contributions, particularly those from Drs. Keisuke Kuida, Alex Kuan, Derek Yang and Stephane Hunot.

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