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Calreticulin is an upstream regulator of calcineurin
BBRC Biochemical and Biophysical Research Communications 311 (2003) 1173–1179 www.elsevier.com/locate/ybbrc
Calreticulin is an upstream regulator of calcineurin Jeffrey Lynch and Marek Michalak* Canadian Institutes of Health Research Membrane Protein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alta., Canada T6G 2H7 Received 3 July 2003
Abstract Ca2þ is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca2þ . Within the ER lumen are proteins that function as Ca2þ buffers and/or molecular chaperones including calreticulin, a multifunctional Ca2þ -binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca2þ storage capacity in the ER and InsP3 receptor mediated Ca2þ release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca2þ -signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Calreticulin; Calcineurin; Endoplasmic reticulum; Cardiac development; Calcium homeostasis
Ca2þ is a universal signalling molecule that affects diverse cellular functions such as secretion, contraction– relaxation, cell motility, cytoplasmic and mitochondrial metabolism, synthesis, modification, and folding of proteins, gene expression, cell cycle progression, and apoptosis [1]. The endoplasmic reticulum (ER) plays a central role in maintaining intracellular Ca2þ homeostasis [1,2]. Ca2þ is released from the ER via the InsP3 receptor and is taken back up into the ER via a Ca2þ pump known as SERCA [1]. Ca2þ also enters the cytoplasm via channels in the plasma membrane. Importantly, there is efficient and physiologically relevant coupling of extracellular Ca2þ entry and intracellular Ca2þ release mediated by interactions between components of the plasma membrane and the ER [3]. For example, Ca2þ released from the ER activates storeoperated Ca2þ channels in the plasma membrane causing cytoplasmic Ca2þ concentrations to increase rapidly. In the cytoplasm, Ca2þ exerts its effects by binding to Ca2þ -activated proteins. Many of these are protein kinases and phosphatases which modulate transcriptional * Corresponding author. Fax: +1-780-492-0886. E-mail address: [email protected] (M. Michalak).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.040
processes involved in cell proliferation and differentiation, and in organ growth and development. Consequently, Ca2þ release from the ER must be very carefully regulated because of its effects in virtually all areas of cell function. This review will focus on the unexpected discovery that calreticulin, a Ca2þ -binding chaperone that resides in the lumen of the ER, plays a role in the activation of Ca2þ -dependent process in the cytoplasm by signalling via calcineurin, a Ca2þ - and calmodulin-dependent protein phosphatase. These observations highlight the importance of ER-dependent signalling in embryogenesis.
The endoplasmic reticulum and calreticulin The organization of the ER indicates that it has significant functional and structural heterogeneity [4]. Importantly, the ER is localized to areas within the cell where it participates in cellular signalling cascades [1]. The lumen of the ER contains a high concentration of Ca2þ , along with numerous Ca2þ -binding chaperones and other proteins which play diverse roles in virtually
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every aspect of ER function, including protein and lipid synthesis and maintenance of Ca2þ homeostasis [5]. Most Ca2þ in the ER is bound to Ca2þ -binding proteins, but a small fraction of “free” Ca2þ is thought to play a significant role in ER function [5,6]. Calreticulin is an ER chaperone which acts, via the calreticulin/calnexin cycle, in the proper folding of many proteins and glycoproteins [7]. It is also a major Ca2þ binding/buffering protein in the lumen of the ER [8] and, as such, is involved in several of the processes that comprise cellular Ca2þ homeostasis, including Ca2þ storage in the ER [9], Ca2þ release from the ER [9,10], SERCA function [11], and activation of store-operated Ca2þ influx [12–16]. Calreticulin has several distinct structural domains. The N-terminal domain and central, P-domain of calreticulin are responsible for its chaperone (protein folding) activity, whereas the C-terminal, acidic region plays a key role in Ca2þ storage within the ER and in ER retrieval of the protein (Fig. 1) [9]. Given calreticulin’s roles in both protein folding and Ca2þ homeostasis, it is not surprising that calreticulindeficiency is embryonic lethal in mice [10]. The major effects of calreticulin-deficiency are observed in the heart [10], and calreticulin-deficient embryos die at E14.5 from impaired development of the ventricular wall [10]. These findings were somewhat surprising because calreticulin
is only a minor component of mature cardiac tissue [10]. However, subsequent studies showed that calreticulin is highly expressed in embryonic heart where it presumably plays an important role during cardiac growth and differentiation [10,17]. Studies on cells derived from crt= embryos indicate that protein folding in the ER is compromised in the absence of calreticulin, resulting in the accumulation of mis-folded protein and activation of unfolded protein responses [18]. Importantly, agonist-mediated Ca2þ release from the ER is also inhibited in crt= cells, likely because Ca2þ handling in the ER and the folding/trafficking of cell surface receptors are impaired [9,10]. Clearly, the absence of this one chaperone has devastating effects on ER function. Studies with calreticulindeficient cells have also revealed that protein folding and Ca2þ signalling processes in the ER are highly interdependent. For example, protein folding depends on the maintenance of normal cellular Ca2þ homeostasis [6,9,19,20]. Equally, the maintenance of normal Ca2þ homeostasis requires proper folding and trafficking of the cell surface receptors and other proteins involved [9,20]. Since Ca2þ release from the ER is inhibited in the absence of calreticulin, then Ca2þ -dependent signalling pathways must also be impaired. We proposed that impaired Ca2þ /calcineurin-dependent transcriptional
Fig. 1. A putative model of calreticulin. The N- and P-domain of calreticulin are modelled based on the NMR studies of the P-domain of calreticulin and crystallographic studies of calnexin (1JHN). The globular N-domain is shown in blue and extended arm of the P-domain is represented in red. Yellow balls represent the cysteine (Cys88–Cys120), which form a C–C bridge in calreticulin. Putative glucose (substrate?) binding site is indicated. Ca2þ -binding the C-domain is shown as a blue cylinder. The N + P-domain is likely a chaperone region (folding unit) of calreticulin, whereas the C-domain is responsible for Ca2þ storage function in the ER lumen.
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activation might be responsible for the impaired cardiogenesis and embryonic lethality observed in calreticulin-deficient mice [10].
Calcineurin Calcineurin is a highly conserved, Ca2þ /calmodulindependent serine/threonine phosphatase (phosphatase 2B) [21,22]. It is a heterotetramer which contains A and B subunits (CaN-A and CaN-B). CaN-A is the catalytic subunit and binds calmodulin; CaN-B is the regulatory subunit and binds Ca2þ [22]. In isolation, CaN-A has relatively low phosphatase activity but this is greatly enhanced when it is associated with CaN-B [23,24]. Calcineurin is best known for its role in the Ca2þ , NF-AT signalling pathways that are involved in T-cell activation [25]. Specifically, calcineurin-dependent dephosphorylation of NF-AT promotes its nuclear translocation and the subsequent activation of specific genes [25]. The activation of calcineurin occurs in response to sustained Ca2þ release from the ER and Ca2þ influx across the plasma membrane [21,25], but it is rather insensitive to Ca2þ transients, such as those necessary for muscle contraction. One unique aspect of calcineurin biology is its substrate specificity which includes transcriptional factors, specific inhibitors, ion channels, apoptotic molecules, and cytoskeletal proteins [21,25]. Calcineurin also affects some of its substrates via recruitment of adaptor proteins, such as FK506-binding protein-12 (FKBP12) [26]. CaN-A can be divided into multiple distinct functional regions (Fig. 2) [22,27]: (1) the N-terminal region (amino acids 15–24) which modulates CaN-A’s catalytic activity by interacting with CaN-B [28]; (2) the catalytic domain, which has amino acid sequence similarities with other protein phosphatases; (3) the CaN-B-binding
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domain (amino acids 350–370); (4) the calmodulinbinding region (amino acids 390–414); and (5) an autoinhibitory (AI) region (amino acids 469–486) which blocks the catalytic site. CaN-B is 170 amino acids in length, contains four Ca2þ -binding EF-hand motifs (Fig. 2), and has significant homology with calmodulin and troponin [22]. The binding of Ca2þ to CaN-B plays a role in the activation of CaN-A [29], but this may be a structural rather than regulatory role [29] with the interaction between CaN-A and CaN-B alone being necessary for the activation of CaN-A [24]. A constitutively active form of CaN-A has been generated by deletion of its AI domain and a portion (C-terminal) of its calmodulin-binding domain. This protein is referred to throughout this paper as “activated-CaN” (Figs. 2 and 3).
Calcineurin in the heart The role of calcineurin in the NF-AT signalling pathway in many tissues is well established [25]. Recent studies indicate that this pathway is critical in cardiac physiology, pathology, and development [10,30–34]. Mice with a disruption of the NF-ATc gene die in utero because of an inability to develop normal heart valves and septa [35,36]. In other mice, the over-expression of a constitutively active form of NF-AT in the heart causes severe cardiac hypertrophy [30]. NF-AT is, apparently, essential for proper cardiac development and plays a role in cardiac hypertrophy [30,35,37,38]. Transgenic mice expressing activated-CaN in the heart (under the control of the a-MHC promoter) also develop severe cardiac hypertrophy and die from heart failure [30]. This results from calcineurin-dependent activation of the NFAT pathway [30]. The effects of expressing activated-CaN in the heart are blocked by the calcineurin inhibitors cyclosporin A (CsA) and FK506 [39]. Long-term CsA treatment disrupts Ca2þ homeostasis and alters the contractile properties of cardiomyocytes, via effects on SERCA and L-type Ca2þ channels [40]. In vivo, CsA prevents muscle regeneration in response to damage [41] and transplant patients treated with CsA exhibit severe skeletal muscle weakness [42]. Importantly, metabolic toxicities [43] and weight loss have also been associated with CsA treatment [31]. The inhibition of calcineurin likely plays a significant role in all of these pathologies.
Signalling through calreticulin and calcineurin in the heart Fig. 2. A schematic representation of calcineurin A (CaN-A), activated, truncated calcineurin A and calcineurin B (CaN-B). Localization of calcineurin B binding site (CnB), calmodulin binding site (CaM), and inhibitory region (AI) is depicted. Four Ca2þ binding EFhand motifs are indicated in CaN-B.
Calreticulin-deficiency is embryonic lethal in mice because of the development of severe cardiac defects, and calreticulin-deficient cells show inhibited InsP3 -dependent Ca2þ release from the ER and impaired folding
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Fig. 3. Relationship between calreticulin and calcineurin. Calreticulin, in the lumen of the ER, plays a role of Ca2þ -binding/storage chaperone. Ca2þ released from ER and Ca2þ entering via store-operated Ca2þ channel activates calcineurin affecting several Ca2þ -dependent transcriptional pathways during organogenesis. In turn, these transcriptional processes have a major influence on normal tissue development and energy metabolism. Calcineurin may also affect tissue development and energy metabolism directly without activating specific transactional processes. NF-AT, nuclear factor of activated T-cells; TF, transcription factor.
and trafficking of proteins [9,10,20]. At this time, it remains to be determined how the activity of calreticulin, within the lumen of the ER, impacts cardiac development. It is known that Ca2þ plays a role in many transcriptional processes, including transcriptional regulation of cardiac specific proteins [44,45]. Since agonistinduced Ca2þ release is inhibited in calreticulin-deficient cells, we proposed that Ca2þ -dependent transcriptional processes may be impaired during early cardiac development in the absence of calreticulin [10]. As already stated, Ca2þ /calcineurin-dependent transcriptional pathways are particularly well understood and are known to play a key role in cardiac physiology [30,44,45]. It is likely that similar transcriptional pathways are critical for early cardiac development, and this suggests a possible relationship between the effects of calreticulin in the ER and the function of calcineurin in the cytosol. Indeed, NF-AT nuclear translocation is compromised in calreticulin-deficient cells, implying that calcineurin activity is also impaired [10]. To investigate the functional relationship between calreticulin and calcineurin in cardiac tissue, we created calreticulin-deficient mice which express activated-CaN in the heart [34]. In the early stages of cardiac development, calreticulin is probably required to ensure normal Ca2þ release from the ER and thus proper activation of calcineurin and its associated transcriptional pathways. However, over-expression of activated-CaN renders calcineurin-dependent pathways less dependent on sustained elevation of cytoplasmic Ca2þ and so calcineurin-dependent pathways should be active even in the absence of Ca2þ release from the ER. Cardiac-specific over-expression of activated-CaN was achieved using an a-MHC promoter [30]. Although
this produces severe cardiac hypertrophy, the mice are fertile and so can be used to breed with calreticulin heterozygote (crtþ= ) mice [34]. We found that over-expression of activated-CaN in the heart was sufficient to rescue calreticulin-deficient embryonic lethality, producing viable calreticulin-deficient mice [34]. In fact, the expression of activated-CaN in the cardiac tissue of these animals restored normal ventricular development [34]. Histological analysis of cardiac tissue from calreticulin-deficient embryos reveals deep intertrabecular recesses and increased fenestration associated with a thinner ventricular wall [10]. In sharp contrast, calreticulin-deficient embryos and newborn mice expressing activated-CaN in the heart do not show these defects in cardiac development [34]. Instead they exhibit normal development of the ventricular wall, with signs of early hypertrophy which probably result from the expression of activated calcineurin [34]. These observations provide a clear molecular explanation for the embryonic lethality seen in calreticulin-deficient mice and indicate that calreticulin is a key upstream player in the Ca2þ -signalling cascades which regulate calcineurin activity. The remarkable reversal of the embryonic lethality that results from calreticulin-deficiency, by the expression of only one protein in the heart (activated calcineurin), highlights the importance of both calreticulin and calcineurin in Ca2þ -dependent signalling cascades during early cardiac development. In the absence of calreticulin, expression of activated-CaN in the heart permits proper progression of cardiac development during embryogenesis. This supports our earlier hypothesis that Ca2þ -dependent, cardiac-specific transcriptional processes are impaired in the absence of calreticulin [10]. Studies on calreticulin-deficient stem
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cells provide further evidence for this hypothesis [46]. Specifically, cardiomyocytes derived from calreticulin-deficient heart have severely disrupted myofibrillogenesis and impaired nuclear translocation of the transcription factor Mef2c [46]. Combined, these findings show that calreticulin and calcineurin play important roles in the Ca2þ -dependent pathways that are essential for myofibrillogenesis and normal cardiac development (Fig. 3). At present the molecular details underlying this are not understood.
Calreticulin/calcineurin signalling outside the heart Although cardiac expression of activated-CaN reversed the embryonic lethality seen in calreticulin-deficient mice, the mice did exhibit severe postnatal pathology and died 7–35 days after birth [34]. The rescue mice expressing calcineurin only in the heart have severe growth retardation and significant metabolic problems [34]. Analysis of sugar in the blood indicates that the mice are hypoglycemic from as early as the first week of postnatal life and the “milky” appearance of their blood serum indicates abnormal lipid metabolism [34]. Specifically, the rescue mice have elevated levels of both cholesterol (over 4-fold) and triacylglycerols (over 9fold) [34]. The underlying cause of the metabolic aberrations in rescue mice is not understood, but they suggest that calreticulin function in the ER is important for normal energy metabolism following birth. Calcineurin may be involved in these calreticulin-dependent pathways in other tissues, as it is in the heart, but the role of calcineurin in metabolism is still largely unexplored. Indirect evidence of its involvement comes from observations made in transplant patients treated with CsA, which is a calcineurin inhibitor. Specifically, these patients often exhibit elevated serum cholesterol and triglyceride levels [43,47,48]. Thus, in calreticulin-deficient mice, serum cholesterol may be elevated because of compromised Ca2þ release from the ER which, in turn, compromises the activation of calcineurin. Calreticulin-deficiency compromises all aspects of ER function, including Ca2þ homeostasis, protein synthesis, and lipid synthesis, and also compromises events outside the ER, including the regulation of calcineurin-dependent pathways (Fig. 3). Another event which might be compromised by calreticulin-deficiency is the action of sterol regulatory element-binding proteins (SREBPs). SREBPs are integral membrane proteins in the ER, but their cytoplasmic NH2 -terminal can be cleaved by a sterol-regulated protease [49]. The cleaved portion of the proteins then dimerizes and the dimer translocates to the nucleus to bind to the sterol regulatory elements of genes that encode proteins necessary for lipid biosynthesis and lipid uptake [49]. When ER function is impacted by
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calreticulin-deficiency, it is possible that SREBP function is also impacted, thereby altering lipid metabolism. Overall, it is likely that the elevated serum triacylglycerol and cholesterol levels observed in calreticulin-deficient rescue mice result from the impairment of several processes, including ER Ca2þ homeostasis, SREBP activity, and calcineurin-dependent signalling pathways. Calreticulin-deficient rescue mice have very low levels of blood glucose [34] and there is no explanation for this observation at present. Calreticulin may play a role in the synthesis, maturation, and secretion of insulin and/or insulin receptors. It could also affect the synthesis and/or targeting of glucose transporters in the gastrointestinal tract, resulting in reduced glucose uptake from the diet. Alternatively, calreticulin-deficiency could affect glucose metabolism via calcineurin-dependent signalling pathways. It is well-established that Ca2þ is of critical importance in hormone secretion from the b-cells of the pancreas [50]. Further, calcineurin inhibitors affect multiple aspects of insulin secretion [51] and the clinical use of FK506 has been associated with development of insulindependent diabetes mellitus [43,47,48]. FK506 is a strong immunosuppressant and one of its main adverse side effects is hyperglycemia [47,52]. The expression of calreticulin is tightly regulated in many tissues. For example, it is highly expressed in the embryonic heart and brain, but is down-regulated after birth [10]. In contrast, in mature organisms calreticulin is highly expressed in the pancreas and liver, tissues that are involved in protein synthesis and secretion, and in the regulation of energy metabolism. Studies with calreticulin-deficient rescue mice indicate that calcineurin-dependent pathways are involved in the effects of calreticulin-deficiency on cardiac development, and may be involved in the effects on metabolism (Fig. 3). Calreticulin is an integral and essential component in maintenance of cellular Ca2þ homeostasis, and as such it appears to be a key upstream regulator of calcineurin function (Fig. 3). Overall, the findings reviewed here underscore the importance of the ER and Ca2þ -dependent signalling pathways during the early stages of cellular commitment and in tissue development during organogenesis.
Acknowledgments This work was supported by the Canadian Institute of Health Research. I.A. is a recipient of a Studentship Award from the HSFC. M.M. is a Canadian Institutes of Health Research Senior Investigator and an Alberta Heritage Foundation for Medical Research Medical Scientist.
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Calreticulin is an upstream regulator of calcineurin Jeffrey Lynch and Marek Michalak* Canadian Institutes of Health Research Membrane Protein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alta., Canada T6G 2H7 Received 3 July 2003
Abstract Ca2þ is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca2þ . Within the ER lumen are proteins that function as Ca2þ buffers and/or molecular chaperones including calreticulin, a multifunctional Ca2þ -binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca2þ storage capacity in the ER and InsP3 receptor mediated Ca2þ release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca2þ -signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Calreticulin; Calcineurin; Endoplasmic reticulum; Cardiac development; Calcium homeostasis
Ca2þ is a universal signalling molecule that affects diverse cellular functions such as secretion, contraction– relaxation, cell motility, cytoplasmic and mitochondrial metabolism, synthesis, modification, and folding of proteins, gene expression, cell cycle progression, and apoptosis [1]. The endoplasmic reticulum (ER) plays a central role in maintaining intracellular Ca2þ homeostasis [1,2]. Ca2þ is released from the ER via the InsP3 receptor and is taken back up into the ER via a Ca2þ pump known as SERCA [1]. Ca2þ also enters the cytoplasm via channels in the plasma membrane. Importantly, there is efficient and physiologically relevant coupling of extracellular Ca2þ entry and intracellular Ca2þ release mediated by interactions between components of the plasma membrane and the ER [3]. For example, Ca2þ released from the ER activates storeoperated Ca2þ channels in the plasma membrane causing cytoplasmic Ca2þ concentrations to increase rapidly. In the cytoplasm, Ca2þ exerts its effects by binding to Ca2þ -activated proteins. Many of these are protein kinases and phosphatases which modulate transcriptional * Corresponding author. Fax: +1-780-492-0886. E-mail address: [email protected] (M. Michalak).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.040
processes involved in cell proliferation and differentiation, and in organ growth and development. Consequently, Ca2þ release from the ER must be very carefully regulated because of its effects in virtually all areas of cell function. This review will focus on the unexpected discovery that calreticulin, a Ca2þ -binding chaperone that resides in the lumen of the ER, plays a role in the activation of Ca2þ -dependent process in the cytoplasm by signalling via calcineurin, a Ca2þ - and calmodulin-dependent protein phosphatase. These observations highlight the importance of ER-dependent signalling in embryogenesis.
The endoplasmic reticulum and calreticulin The organization of the ER indicates that it has significant functional and structural heterogeneity [4]. Importantly, the ER is localized to areas within the cell where it participates in cellular signalling cascades [1]. The lumen of the ER contains a high concentration of Ca2þ , along with numerous Ca2þ -binding chaperones and other proteins which play diverse roles in virtually
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every aspect of ER function, including protein and lipid synthesis and maintenance of Ca2þ homeostasis [5]. Most Ca2þ in the ER is bound to Ca2þ -binding proteins, but a small fraction of “free” Ca2þ is thought to play a significant role in ER function [5,6]. Calreticulin is an ER chaperone which acts, via the calreticulin/calnexin cycle, in the proper folding of many proteins and glycoproteins [7]. It is also a major Ca2þ binding/buffering protein in the lumen of the ER [8] and, as such, is involved in several of the processes that comprise cellular Ca2þ homeostasis, including Ca2þ storage in the ER [9], Ca2þ release from the ER [9,10], SERCA function [11], and activation of store-operated Ca2þ influx [12–16]. Calreticulin has several distinct structural domains. The N-terminal domain and central, P-domain of calreticulin are responsible for its chaperone (protein folding) activity, whereas the C-terminal, acidic region plays a key role in Ca2þ storage within the ER and in ER retrieval of the protein (Fig. 1) [9]. Given calreticulin’s roles in both protein folding and Ca2þ homeostasis, it is not surprising that calreticulindeficiency is embryonic lethal in mice [10]. The major effects of calreticulin-deficiency are observed in the heart [10], and calreticulin-deficient embryos die at E14.5 from impaired development of the ventricular wall [10]. These findings were somewhat surprising because calreticulin
is only a minor component of mature cardiac tissue [10]. However, subsequent studies showed that calreticulin is highly expressed in embryonic heart where it presumably plays an important role during cardiac growth and differentiation [10,17]. Studies on cells derived from crt= embryos indicate that protein folding in the ER is compromised in the absence of calreticulin, resulting in the accumulation of mis-folded protein and activation of unfolded protein responses [18]. Importantly, agonist-mediated Ca2þ release from the ER is also inhibited in crt= cells, likely because Ca2þ handling in the ER and the folding/trafficking of cell surface receptors are impaired [9,10]. Clearly, the absence of this one chaperone has devastating effects on ER function. Studies with calreticulindeficient cells have also revealed that protein folding and Ca2þ signalling processes in the ER are highly interdependent. For example, protein folding depends on the maintenance of normal cellular Ca2þ homeostasis [6,9,19,20]. Equally, the maintenance of normal Ca2þ homeostasis requires proper folding and trafficking of the cell surface receptors and other proteins involved [9,20]. Since Ca2þ release from the ER is inhibited in the absence of calreticulin, then Ca2þ -dependent signalling pathways must also be impaired. We proposed that impaired Ca2þ /calcineurin-dependent transcriptional
Fig. 1. A putative model of calreticulin. The N- and P-domain of calreticulin are modelled based on the NMR studies of the P-domain of calreticulin and crystallographic studies of calnexin (1JHN). The globular N-domain is shown in blue and extended arm of the P-domain is represented in red. Yellow balls represent the cysteine (Cys88–Cys120), which form a C–C bridge in calreticulin. Putative glucose (substrate?) binding site is indicated. Ca2þ -binding the C-domain is shown as a blue cylinder. The N + P-domain is likely a chaperone region (folding unit) of calreticulin, whereas the C-domain is responsible for Ca2þ storage function in the ER lumen.
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activation might be responsible for the impaired cardiogenesis and embryonic lethality observed in calreticulin-deficient mice [10].
Calcineurin Calcineurin is a highly conserved, Ca2þ /calmodulindependent serine/threonine phosphatase (phosphatase 2B) [21,22]. It is a heterotetramer which contains A and B subunits (CaN-A and CaN-B). CaN-A is the catalytic subunit and binds calmodulin; CaN-B is the regulatory subunit and binds Ca2þ [22]. In isolation, CaN-A has relatively low phosphatase activity but this is greatly enhanced when it is associated with CaN-B [23,24]. Calcineurin is best known for its role in the Ca2þ , NF-AT signalling pathways that are involved in T-cell activation [25]. Specifically, calcineurin-dependent dephosphorylation of NF-AT promotes its nuclear translocation and the subsequent activation of specific genes [25]. The activation of calcineurin occurs in response to sustained Ca2þ release from the ER and Ca2þ influx across the plasma membrane [21,25], but it is rather insensitive to Ca2þ transients, such as those necessary for muscle contraction. One unique aspect of calcineurin biology is its substrate specificity which includes transcriptional factors, specific inhibitors, ion channels, apoptotic molecules, and cytoskeletal proteins [21,25]. Calcineurin also affects some of its substrates via recruitment of adaptor proteins, such as FK506-binding protein-12 (FKBP12) [26]. CaN-A can be divided into multiple distinct functional regions (Fig. 2) [22,27]: (1) the N-terminal region (amino acids 15–24) which modulates CaN-A’s catalytic activity by interacting with CaN-B [28]; (2) the catalytic domain, which has amino acid sequence similarities with other protein phosphatases; (3) the CaN-B-binding
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domain (amino acids 350–370); (4) the calmodulinbinding region (amino acids 390–414); and (5) an autoinhibitory (AI) region (amino acids 469–486) which blocks the catalytic site. CaN-B is 170 amino acids in length, contains four Ca2þ -binding EF-hand motifs (Fig. 2), and has significant homology with calmodulin and troponin [22]. The binding of Ca2þ to CaN-B plays a role in the activation of CaN-A [29], but this may be a structural rather than regulatory role [29] with the interaction between CaN-A and CaN-B alone being necessary for the activation of CaN-A [24]. A constitutively active form of CaN-A has been generated by deletion of its AI domain and a portion (C-terminal) of its calmodulin-binding domain. This protein is referred to throughout this paper as “activated-CaN” (Figs. 2 and 3).
Calcineurin in the heart The role of calcineurin in the NF-AT signalling pathway in many tissues is well established [25]. Recent studies indicate that this pathway is critical in cardiac physiology, pathology, and development [10,30–34]. Mice with a disruption of the NF-ATc gene die in utero because of an inability to develop normal heart valves and septa [35,36]. In other mice, the over-expression of a constitutively active form of NF-AT in the heart causes severe cardiac hypertrophy [30]. NF-AT is, apparently, essential for proper cardiac development and plays a role in cardiac hypertrophy [30,35,37,38]. Transgenic mice expressing activated-CaN in the heart (under the control of the a-MHC promoter) also develop severe cardiac hypertrophy and die from heart failure [30]. This results from calcineurin-dependent activation of the NFAT pathway [30]. The effects of expressing activated-CaN in the heart are blocked by the calcineurin inhibitors cyclosporin A (CsA) and FK506 [39]. Long-term CsA treatment disrupts Ca2þ homeostasis and alters the contractile properties of cardiomyocytes, via effects on SERCA and L-type Ca2þ channels [40]. In vivo, CsA prevents muscle regeneration in response to damage [41] and transplant patients treated with CsA exhibit severe skeletal muscle weakness [42]. Importantly, metabolic toxicities [43] and weight loss have also been associated with CsA treatment [31]. The inhibition of calcineurin likely plays a significant role in all of these pathologies.
Signalling through calreticulin and calcineurin in the heart Fig. 2. A schematic representation of calcineurin A (CaN-A), activated, truncated calcineurin A and calcineurin B (CaN-B). Localization of calcineurin B binding site (CnB), calmodulin binding site (CaM), and inhibitory region (AI) is depicted. Four Ca2þ binding EFhand motifs are indicated in CaN-B.
Calreticulin-deficiency is embryonic lethal in mice because of the development of severe cardiac defects, and calreticulin-deficient cells show inhibited InsP3 -dependent Ca2þ release from the ER and impaired folding
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Fig. 3. Relationship between calreticulin and calcineurin. Calreticulin, in the lumen of the ER, plays a role of Ca2þ -binding/storage chaperone. Ca2þ released from ER and Ca2þ entering via store-operated Ca2þ channel activates calcineurin affecting several Ca2þ -dependent transcriptional pathways during organogenesis. In turn, these transcriptional processes have a major influence on normal tissue development and energy metabolism. Calcineurin may also affect tissue development and energy metabolism directly without activating specific transactional processes. NF-AT, nuclear factor of activated T-cells; TF, transcription factor.
and trafficking of proteins [9,10,20]. At this time, it remains to be determined how the activity of calreticulin, within the lumen of the ER, impacts cardiac development. It is known that Ca2þ plays a role in many transcriptional processes, including transcriptional regulation of cardiac specific proteins [44,45]. Since agonistinduced Ca2þ release is inhibited in calreticulin-deficient cells, we proposed that Ca2þ -dependent transcriptional processes may be impaired during early cardiac development in the absence of calreticulin [10]. As already stated, Ca2þ /calcineurin-dependent transcriptional pathways are particularly well understood and are known to play a key role in cardiac physiology [30,44,45]. It is likely that similar transcriptional pathways are critical for early cardiac development, and this suggests a possible relationship between the effects of calreticulin in the ER and the function of calcineurin in the cytosol. Indeed, NF-AT nuclear translocation is compromised in calreticulin-deficient cells, implying that calcineurin activity is also impaired [10]. To investigate the functional relationship between calreticulin and calcineurin in cardiac tissue, we created calreticulin-deficient mice which express activated-CaN in the heart [34]. In the early stages of cardiac development, calreticulin is probably required to ensure normal Ca2þ release from the ER and thus proper activation of calcineurin and its associated transcriptional pathways. However, over-expression of activated-CaN renders calcineurin-dependent pathways less dependent on sustained elevation of cytoplasmic Ca2þ and so calcineurin-dependent pathways should be active even in the absence of Ca2þ release from the ER. Cardiac-specific over-expression of activated-CaN was achieved using an a-MHC promoter [30]. Although
this produces severe cardiac hypertrophy, the mice are fertile and so can be used to breed with calreticulin heterozygote (crtþ= ) mice [34]. We found that over-expression of activated-CaN in the heart was sufficient to rescue calreticulin-deficient embryonic lethality, producing viable calreticulin-deficient mice [34]. In fact, the expression of activated-CaN in the cardiac tissue of these animals restored normal ventricular development [34]. Histological analysis of cardiac tissue from calreticulin-deficient embryos reveals deep intertrabecular recesses and increased fenestration associated with a thinner ventricular wall [10]. In sharp contrast, calreticulin-deficient embryos and newborn mice expressing activated-CaN in the heart do not show these defects in cardiac development [34]. Instead they exhibit normal development of the ventricular wall, with signs of early hypertrophy which probably result from the expression of activated calcineurin [34]. These observations provide a clear molecular explanation for the embryonic lethality seen in calreticulin-deficient mice and indicate that calreticulin is a key upstream player in the Ca2þ -signalling cascades which regulate calcineurin activity. The remarkable reversal of the embryonic lethality that results from calreticulin-deficiency, by the expression of only one protein in the heart (activated calcineurin), highlights the importance of both calreticulin and calcineurin in Ca2þ -dependent signalling cascades during early cardiac development. In the absence of calreticulin, expression of activated-CaN in the heart permits proper progression of cardiac development during embryogenesis. This supports our earlier hypothesis that Ca2þ -dependent, cardiac-specific transcriptional processes are impaired in the absence of calreticulin [10]. Studies on calreticulin-deficient stem
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cells provide further evidence for this hypothesis [46]. Specifically, cardiomyocytes derived from calreticulin-deficient heart have severely disrupted myofibrillogenesis and impaired nuclear translocation of the transcription factor Mef2c [46]. Combined, these findings show that calreticulin and calcineurin play important roles in the Ca2þ -dependent pathways that are essential for myofibrillogenesis and normal cardiac development (Fig. 3). At present the molecular details underlying this are not understood.
Calreticulin/calcineurin signalling outside the heart Although cardiac expression of activated-CaN reversed the embryonic lethality seen in calreticulin-deficient mice, the mice did exhibit severe postnatal pathology and died 7–35 days after birth [34]. The rescue mice expressing calcineurin only in the heart have severe growth retardation and significant metabolic problems [34]. Analysis of sugar in the blood indicates that the mice are hypoglycemic from as early as the first week of postnatal life and the “milky” appearance of their blood serum indicates abnormal lipid metabolism [34]. Specifically, the rescue mice have elevated levels of both cholesterol (over 4-fold) and triacylglycerols (over 9fold) [34]. The underlying cause of the metabolic aberrations in rescue mice is not understood, but they suggest that calreticulin function in the ER is important for normal energy metabolism following birth. Calcineurin may be involved in these calreticulin-dependent pathways in other tissues, as it is in the heart, but the role of calcineurin in metabolism is still largely unexplored. Indirect evidence of its involvement comes from observations made in transplant patients treated with CsA, which is a calcineurin inhibitor. Specifically, these patients often exhibit elevated serum cholesterol and triglyceride levels [43,47,48]. Thus, in calreticulin-deficient mice, serum cholesterol may be elevated because of compromised Ca2þ release from the ER which, in turn, compromises the activation of calcineurin. Calreticulin-deficiency compromises all aspects of ER function, including Ca2þ homeostasis, protein synthesis, and lipid synthesis, and also compromises events outside the ER, including the regulation of calcineurin-dependent pathways (Fig. 3). Another event which might be compromised by calreticulin-deficiency is the action of sterol regulatory element-binding proteins (SREBPs). SREBPs are integral membrane proteins in the ER, but their cytoplasmic NH2 -terminal can be cleaved by a sterol-regulated protease [49]. The cleaved portion of the proteins then dimerizes and the dimer translocates to the nucleus to bind to the sterol regulatory elements of genes that encode proteins necessary for lipid biosynthesis and lipid uptake [49]. When ER function is impacted by
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calreticulin-deficiency, it is possible that SREBP function is also impacted, thereby altering lipid metabolism. Overall, it is likely that the elevated serum triacylglycerol and cholesterol levels observed in calreticulin-deficient rescue mice result from the impairment of several processes, including ER Ca2þ homeostasis, SREBP activity, and calcineurin-dependent signalling pathways. Calreticulin-deficient rescue mice have very low levels of blood glucose [34] and there is no explanation for this observation at present. Calreticulin may play a role in the synthesis, maturation, and secretion of insulin and/or insulin receptors. It could also affect the synthesis and/or targeting of glucose transporters in the gastrointestinal tract, resulting in reduced glucose uptake from the diet. Alternatively, calreticulin-deficiency could affect glucose metabolism via calcineurin-dependent signalling pathways. It is well-established that Ca2þ is of critical importance in hormone secretion from the b-cells of the pancreas [50]. Further, calcineurin inhibitors affect multiple aspects of insulin secretion [51] and the clinical use of FK506 has been associated with development of insulindependent diabetes mellitus [43,47,48]. FK506 is a strong immunosuppressant and one of its main adverse side effects is hyperglycemia [47,52]. The expression of calreticulin is tightly regulated in many tissues. For example, it is highly expressed in the embryonic heart and brain, but is down-regulated after birth [10]. In contrast, in mature organisms calreticulin is highly expressed in the pancreas and liver, tissues that are involved in protein synthesis and secretion, and in the regulation of energy metabolism. Studies with calreticulin-deficient rescue mice indicate that calcineurin-dependent pathways are involved in the effects of calreticulin-deficiency on cardiac development, and may be involved in the effects on metabolism (Fig. 3). Calreticulin is an integral and essential component in maintenance of cellular Ca2þ homeostasis, and as such it appears to be a key upstream regulator of calcineurin function (Fig. 3). Overall, the findings reviewed here underscore the importance of the ER and Ca2þ -dependent signalling pathways during the early stages of cellular commitment and in tissue development during organogenesis.
Acknowledgments This work was supported by the Canadian Institute of Health Research. I.A. is a recipient of a Studentship Award from the HSFC. M.M. is a Canadian Institutes of Health Research Senior Investigator and an Alberta Heritage Foundation for Medical Research Medical Scientist.
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