The Critical Role of Calpain in Cell Proliferation

Calpain was first described as a neutral, calcium-activated proteinase in the soluble fraction of rat brain [1]. It accomplishes its proteolytic activity in the cytoplasm, not in the lysosomes at a neutral pH. Calpain was named calcium-activated neutral protease (CANP) after purification from chicken skeletal muscle in 1978 [2]. In 1984, Ohno et al. [3] found that the calpain is a fusion gene product with a combination of papain-like cysteine protease and calmodulin-like calcium-binding domains. The members of calpain superfamily can be found in many different species from Homo sapiens to the lower organism including invertebrates, plants, fungi, yeasts and bacteria. In mammalian cells, there are 15 genes encoding the large catalytic subunits, and two genes encoding small regulatory subunits (Table 1) [4,5]. They can be classified according to their localization (ubiquitous or tissue-specific). Several calpain isoforms are ubiquitously expressed in the cytosol (calpain 1, 2, 5, 7, 10, 13, 14, 15 and 16). The others show tissue-specific expression pattern (calpain 3, 6, 8, 9, 11 and 12). For example, calpain 3 is skeletal muscle specific [6], calpain 8 is specific to stomach smooth muscle [7], and the digestive track contains calpain 9 [8,9]. Based on their domain structure calpain can be divided into two classes (typical or atypical). Typical calpain (1, 2, 3, 8, 9, 11, 12, 13 and 14) have four domains in their 80 kDa large subunit as well as encode calmodulin-like EF-hands in domain IV. In contrast, atypical calpain (5, 6, 7, 10, 15 and 16) do not have EF-hands in domain IV and in some domains have been deleted or replaced [5,10]. The calpain 1, 2 and 9 form heterodimer with the 30 kDa subunit, while the other typical calpain (calpain 3, 8, 11, 12 and 14) do not dimerize with the small regulatory subunit, although they have domain IV. The atypical calpain are unsuitable for dimerization due to lack of domain IV.


Introduction
Calpain was first described as a neutral, calcium-activated proteinase in the soluble fraction of rat brain [1]. It accomplishes its proteolytic activity in the cytoplasm, not in the lysosomes at a neutral pH. Calpain was named calcium-activated neutral protease (CANP) after purification from chicken skeletal muscle in 1978 [2]. In 1984, Ohno et al. [3] found that the calpain is a fusion gene product with a combination of papain-like cysteine protease and calmodulin-like calcium-binding domains. The members of calpain superfamily can be found in many different species from Homo sapiens to the lower organism including invertebrates, plants, fungi, yeasts and bacteria. In mammalian cells, there are 15 genes encoding the large catalytic subunits, and two genes encoding small regulatory subunits (Table 1) [4,5]. They can be classified according to their localization (ubiquitous or tissue-specific). Several calpain isoforms are ubiquitously expressed in the cytosol (calpain 1, 2, 5, 7, 10, 13, 14, 15 and 16). The others show tissue-specific expression pattern (calpain 3, 6, 8, 9, 11 and 12). For example, calpain 3 is skeletal muscle specific [6], calpain 8 is specific to stomach smooth muscle [7], and the digestive track contains calpain 9 [8,9]. Based on their domain structure calpain can be divided into two classes (typical or atypical). Typical calpain (1, 2, 3, 8, 9, 11, 12, 13 and 14) have four domains in their 80 kDa large subunit as well as encode calmodulin-like EF-hands in domain IV. In contrast, atypical calpain (5, 6, 7, 10, 15 and 16) do not have EF-hands in domain IV and in some domains have been deleted or replaced [5,10]. The calpain 1, 2 and 9 form heterodimer with the 30 kDa subunit, while the other typical calpain (calpain 3, 8, 11, 12 and 14) do not dimerize with the small regulatory subunit, although they have domain IV. The atypical calpain are unsuitable for dimerization due to lack of domain IV.

Structure
The most intensely studied members of the calpain superfamily are the mammalian ubiquitous calpain 1 and 2. They were named according to their Ca 2+ requirement for activation in vitro. Calpain 1 and 2 need micro-molar and milli-molar Ca 2+ concentrations, respectively, for their proteolytic activity [11,12]. They are heterodimers and consist of a distinct 80 kDa large catalytic subunit (encoded by CAPN1 and CAPN2, respectively) and a common 30 kDa small subunit (encoded by CAPNS1) that regulates calpain activity [13,14]. The catalytic subunit can be further divided into four functional domains (DI-DIV), while the small subunit comprises two domains (DV-DVI) ( Figure 1) [15,16]. Domain I is the N-terminal region of the large subunit that can interact with the DVI of the small subunit and stabilize the protein. It contains 19 amino acid residues that can be cleaved by autolysis during activation. The protease domain (DII) contains the active site of catalytic triad (Cys105, His262 and Asn286). Like other cysteine proteases, it has two subdomains, IIa and IIb. In the inactive state (in the absence of Ca 2+ ), the Cys105 residue is located on the subdomain IIa and the His262/ Asn286 residues are on the subdomain IIb. Thus, they are far from each other to form the catalytic site. Interactions between DI and DIIa as well as DIII and DIIb help maintain this inactive structure. It has been proposed that calcium-induced conformational changes lead to realign the key residues and create the functional active site [17]. Domain III has a β-sandwich structure which is similar to the C2 domain. C2 domain can be found in proteinkinase C and phospholipase C, which plays a role in the calcium and phospholipids binding of the proteins [18]. This domain can bind phospholipids in calcium dependent manner and may have a role in the activation of the enzyme [19]. Domain IV and domain VI are the C-terminal ends of the catalytic and regulatory subunits, respectively, and both of them contain five EFhand motifs. The first four EF-hands are responsible for the calcium binding and show structural similarity to the calmodulin. The fifth EF-hand contribute to the heterodimerization of the subunits [20][21][22]. Domain V is the amino-terminal end of the regulatory subunit and contains clusters of glycine residues. Because of its hydrophobic feature it has an important role in the membrane anchoring [23]. Recently, a second regulatory subunit has been described, but its physiological function is not revealed yet [24].

Regulation and activation
The micro-molar and milli-molar Ca 2+ concentrations required for calpain 1 and 2 in vitro activation are far above the Ca 2+ level available in the cytoplasm. Therefore numerous factors have been proposed that can reduce this enormous calcium requirement including autoproteolytic cleavage of the N-terminal tail, phospholipid binding, posttranslational modification (e.g. phosphorylation), endogenous inhibitor calpastatin and binding to regulator proteins.
The native calpain exists as an inactive proenzyme in the cytosol and cuts its N-terminal part of both subunits upon activation causing the dissociation of the catalytic and regulatory subunits most of the cases [12,[39][40][41]. The autolysis of the large and small subunits occurs rapidly in two and three stages, respectively [42][43][44]. During this process the enzyme becomes active and has a lower Ca 2+ requirement for its activation [45][46][47].
Several phospholipids (e.g. phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate) have important roles in the regulation of the calpain by reducing the Ca 2+ concentration required for their in vitro activation [19,48,49]. Increase in intracellular Ca 2+ level can evoke the translocation of the calpain from the cytosol to the plasma membrane, where it can interact with phospholipids and become catalytically active [40]. Two phospholipids binding sites have been identified in the N-terminal region of the small subunit and the domain III of the large subunit, respectively [19,50].
Phosphorylation also takes part in controlling calpain activity. Earlier report suggests that the calpain could not be phosphorylated in vivo [51], but nine and eight phosphorylated residues were later identified in calpain 1 and 2, respectively [4]. This posttranslational modification is a double-edged sword because it can up-regulate and down-regulate calpain activity. Calpain 2 can be directly phosphorylated at Ser50 by the extracellular signal-regulated protein kinase (ERK) after epidermal growth factor (EGF) stimulation causing the activation of the enzyme independently of calcium [52,53]. In contrast, phosphorylation of the calpain 2 at Ser369/370 by protein kinase A (PKA) blocks the EGFRstimulated calpain activation by freezing the enzyme in an inactive conformation [54,55]. Interestingly, the muscle-specific calpain 3 (p94) has a glutamic acid at Ser50 site, in which the negative charge mimics the effect of phosphorylation, and does not need increased intracellular calcium level for its activation [56]. Calpain 1 does not contain phoshorylatable amino acid residue at this position and it was revealed that it can be activated by cytokines in calcium flux dependent manner [57]. Nicotine induces phosphorylation of calpain 1 and 2 by protein kinase Cι which is associated with elevated activation and secretion of the enzymes [58]. Recent observation indicates that the closely related calpain homologue, calpain B can also be regulated by EGF-stimulated phosphorylation in fruit flies [59], which further emphasizes the regulatory role of this posttranslational modification in the calpain system. More recently it was found that the ERK-and PKA kinases-mediated phosphorylation of the calpain 2 regulates the distribution of the enzyme between cytoplasm and plasma membrane, indirectly contributing to its activation [60].  [67][68][69]. Calpastatin can interact with both native and autolyzed form of calpain, although its affinity to the autolyzed calpain is higher [70,71]. Phosphorylation of calpastatin by PKA can modify its subcellular localization and inhibitory specificity [72].
Several proteins were identified as calpain activators which can reduce the calcium requirement for calpain activation and facilitates the autolysis [73,74]. Some of them are calpain 1 specific [75,76], while others are specific for calpain 2 [77][78][79]. But their precise role is poorly investigated.

Calpain in cell proliferation
Calpain 1 and calpain 2 are the best characterized typical calpain isoforms which have been shown to be involved in many basic cellular processes such as cell proliferation and differentiation in various mammalian cell types. The role of calpain in cell proliferation was revealed in some earlier experiments using calpain inhibitors. Calpain inhibitor calpeptin (benzyloxycarbonyl-Leu-nLeu-H) and other thiol protease inhibitors were shown to reversibly inhibit the PDGF-BB-as well as serum-induced bovine aortic smooth muscle cell (BASMC) cycle progression in vitro [80]. Ariyoshi et al. [81] found that the cellpermeable calpeptin and its analogue (benzyloxycarbonyl-Leu-Met-H), as well as antisense oligonucleotide against calpain 2 (m-calpain) block the proliferation of vascular smooth muscle cell (VSMC) in dose-dependent manner supporting the fact that the calpain activity is involved in this process. Similar findings were observed in other types of cells. Culturing of the Chinese hamster ovary cell line (CHO) in the presence of cell-permeant calpain inhibitor ZLLY-CHN2 (benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone) causes not only a decrease in calpain 1 (µ-calpain) protein level but it also diminishes the growth rate of the cells [82]. Depletion of the calpain small subunit (calpain 4) using specific antisense oligonucleotide inhibits the proliferation of WI-38 human fibroblasts and HeLa cells [83]. Zhang et al. [84] also reported that the calpain-selective inhibitor ZLLY-CHN2 blocks the serum-stimulated growth and cell cycle progression into S-phase in WI-38 human fibroblasts cells. It was shown that overexpression of calpastatin in CHO cell lines significantly decreases the growth of isolated colonies [85]. The pro-proliferative effect of calpain was also revealed in osteoblast cells. It was reported that cellpermeable calpain inhibitor attenuates the proliferation of MC3T3-E1 preosteoblasts cells [86,87], while specific disruption of calpain 4 results in impaired proliferation and differentiation of osteoblastic cells [88]. Taken together, these observations strongly indicate the role of calpain in the proliferation of different mammalian cells.
Several mechanisms are responsible for calpain-mediated cell proliferation. Carragher [89] reported that the calpain-calpastatin system contributes to the v-Src induced cell cycle progression, cell transformation and motility. They found that v-Src activation increases the calpain 2 protein level which is associated with degradation of calpastatin and focal adhesion kinase (FAK). The elevated calpain activity causes disassembly of focal adhesion complexes and initiation of cell motility. Calpain also induces hyperphosphorylation of the retinoblastoma protein (pRb) and increases the level of cell cycle proteins (cyclins D, A and cyclin-dependent kinase 2) facilitating the progression of transformed cells through the G1 stage of the cell cycle. Inhibition of calpain activity by overexpression of calpastatin or using calpain inhibitors attenuates the effect of calpain on the cell proliferation and motility. Other report has indicated that calpain is involved in mitosis. Knockdown of calpain 2 expression, but not calpain 1, using specific siRNA and/or blocking the calpain activity by specific inhibitors cause abnormal mitosis which is accompanied by chromosome misalignment. Moreover, calpain inhibition delays the prometaphase events and suppresses the generation of polar ejection force on chromosomes [90]. Flow-cytometric analysis of calpain inhibition revealed that calpain activity is not only required to promote the cell cycle at the restriction, G1 checkpoint, but also has significant role in the S and G2M phase progression [27]. Furthermore, Ho et al. [91] reported that calpain 2 mediates the proliferation, migration, and tumorigenesis of mammary cancer cells via Akt-FoxO-p27 Kip1 signaling pathway. Silencing of the calpain 2 isoform results in increases in protein phosphatase 2A (PP2A) level and reduction of Akt activation. Impaired Akt activity contributes to the activation and nuclear translocation of FoxO3a transcription factor which is associated with elevated expression level of cyclin-dependent kinase inhibitor p27 Kip1 and a reduction in the breast carcinoma cell proliferation. Finally, our recent study provides strong evidence that calpain governs proliferation and collagen synthesis of pulmonary artery smooth muscle cells (PASMCs) induced by EGF and PDGF. This effect is contributed to calpain-mediated cleavage and activation of intracellular transforming growth factor-β1 (TGF-β1) in PASMCs. Incubation of the PASMCs with EGF and PDGF significantly increases the calpain activity, cell proliferation and collagen synthesis. The effects of the growth factors are attenuated using specific calpain inhibitor MDL28170 or siRNAs against calpain 1 and calpain 2. More importantly we found that conditional knockout of calpain 4 and calpain inhibitor MDL28170 prevent the progression of pulmonary vascular remodeling induced by hypoxia and monocrotaline [92].
Growing body of evidence demonstrates that calpain-mediated cell proliferation is associated to other complex processes such as cell migration, differentiation (e.g. angiogenesis) and survival. Several studies emphasize the role of calpain in regulating the cell migration. It was revealed that calpain mediates the cleavage of focal adhesion kinase (pp125 FAK ), paxillin, and talin, contributing to the focal adhesion disassembly and cell motility. Calpain inhibition attenuates the proteolytic cleavage of the focal adhesion proteins, preventing the dissolution of the focal adhesion complex and cell migration [93,94]. Depletion of the calpain 1 and 2 isoforms using specific siRNAs showed that the calpain 2 activity is responsible for the limited proteolysis of several cytoskeletal and focal adhesion components such as FAK, paxillin, spectrin, and talin. It was also confirmed that calpain 2 isoform limits the membrane projections and transient membrane activity [95]. The same group demonstrated that silencing of calpain 2 not only diminishes the talin proteolysis but also reduced the disassembly rates of adhesion complexes [96]. Calpain is also implicated in the growth factor-induced cell migration. Glading et al. [52,97] found that inhibitions of either calpain activity or ERK signaling pathway by specific inhibitors (calpeptin, calpain inhibitor I or PD98059) abolish the EGF-induced de-adhesion and cell migration. They also determined that the EGF-mediated calpain activation occurs via ERK/MAP kinase dependent phosphorylation and independent of increased intracellular calcium level. Specific knockdown of calpain 2, but not calpain 1, reduces EGF-induced cell motility. Furthermore, it was confirmed that EGF-induced calpain activity requires plasma membrane-localized activation of EGFR and ERK [98]. Interferon inducible protein-10 (IP-10) abrogates the EGF-induced cell migration by blocking the calpain activity through cAMP-dependent PKA phosphorylation. Inhibition of the PKA activation prevents the inhibitory effect of IP-10 on the EGFinduced motility and calpain activation [55,99]. IP-10 also inhibits the VEGF-mediated calpain activation which is associated with reduced endothelial tube formation and cell motility. Down-regulation of the PKA protein level by specific siRNA or using PKA inhibitors reverses the IP-10 inhibition of VEGF-induced calpain activation [100]. The importance of calpain in the growth factor-mediated cell migration is also supported by other studies. The increases in the migration rate of myoblasts by insulin-like growth factor-1 (IGF-1), TGF-β1, and insulin are related to calpain. Incubation of cells with these growth factors elevates the expression level and activity of calpain 2. The calpain inhibitor, calpeptin, completely blocks the calpain activity diminishing the growth factors-induced cell migration [101,102]. It was also shown that incubation of pulmonary artery endothelial cells (PAECs) with cigarette smoke extract (CSE) causes dose-dependent inhibition of calpain activity and cell proliferation. Calpain inhibitor-1 which inhibits both calpain 1 and calpain 2 attenuates the angiogenesis and potentiates the inhibitory effect of CSE. Moreover, depletion of calpastatin with antisense oligodeoxyribonucleotides prevents CSEinduced decreases in calpain activity and angiogenesis. Collectively, these findings indicate that CSE induced inhibition of angiogenesis is mediated by calpain inhibition [103]. Mo et al. [104] revealed that calpain may have a role in the progression of angiogenesis under hypoxic condition. Overexpression of the hypoxia-inducible factor-1α (HIF-1α) in human umbilical vein endothelial cells (HUVECs) elevates the expression of VEGF, Na + /H + exchanger-1 (NHE1) and calpain as well as endothelial cell proliferation, migration and tube formation. Specific siRNA-mediated depletion of NHE1 diminishes the HIF-1αinduced calpain 2 expression and activity as well as angiogenesis. They also found that VEGF prevents the inhibitory effect of siRNA against NHE1, while the calpain inhibitor ALLM abolishes the protective effect of the VEGF. These data suggest that the NHE1 and calpain 2 have a role in the hypoxia-induced cell proliferation. Incubation of human pulmonary microvascular endothelial cells (PMECs) with VEGF increases the activity and protein content of calpain 2. Inhibition of calpain activity using calpain 2 specific siRNA or by overexpression of calpastatin attenuates VEGF-induced increases in angiogenesis, supporting the mediator's role of calpain 2 in endothelial angiogenesis [105,106]. Youn et al. [107] found that calpain mediates the VEGFinduced endothelial nitric oxide (NO) production through the ezrin/ calpain/PI3K/AMPK/AKT/eNOS axis contributing to the growth factor-stimulated angiogenesis. They claimed that VEGF provokes the membrane translocation and activation of calpain in ezrin dependent manner, causing PI3K/AMPK/AKT activation, eNOS phosphorylation at Ser1179 residue and NO production. A recent study shows that a novel calpain inhibitor, SNJ-1945, attenuates the VEGF-stimulated angiogenesis in human retinal microvascular endothelial cells (HRMECs). Treatment of HRMECs with VEGF causes significant increases mainly in calpain 2 activity as well as in endothelial tube formation and cell migration which are diminished in the presence of calpain inhibitor [108]. In addition, calpain plays an important role in the skin wound healing. Transgenic mice that overexpress calpastatin exhibit impaired wound healing and reduced cell proliferation in the epidermis and delayed re-epithelization. The calpain inhibition also decreases collagen I synthesis and blocks the myofibroblast differentiation and the scar formation [109]. Calpain-mediated cell proliferation is associated with cell survival. Depletion of the calpain 1 using specific siRNA causes significant reduction in the viability and proliferation of skeletal muscle satellite cells. Calpain 1 silencing enhances the expression of pro-apoptotic genes and reduces the genes for cell proliferation, differentiation, and migration. These results support that calpain 1 is implicated in controlling the proliferation and survival of satellite cells [110]. Calpain contributes the development of resistance to chemotherapic agents in tumor cell lines. Knockdown of calpain 2 by isoform specific siRNA or calpain inhibitor ALLN increase the protein level of the inhibitory subunit, IκBα, causing significant reduction in NF-κB activation. In addition, down-regulation of calpain 2 significantly resensitizes the anticancer drug resistant cell lines, indicating calpain may function as a resistance mediator during anticancer therapy [111].

Summary
In summary, convincing evidence highlights that the calpain system especially the ubiquitously expressed calpain 1 and 2 are involved in a broad range of physiological processes including cell growth and proliferation in many types of cells. Perturbed expression and activity of calpain 1 and/or 2 play a key role in tumor cell proliferation, migration and invasion as well as angiogenesis contributing to the development of different types of cancer [105,[111][112][113]. Increased calpain activation also mediates pulmonary and systemic vascular remodeling in pulmonary hypertension and angiotensin II-induced hypertension [92,114,115]. On the other hand, calpain is implicated in other pathological states in which cell proliferation is not involved [31][32][33]. For instance, mutations in the calpain 3 encoding gene (CAPN3) are responsible for the limb-girdle muscular dystrophy 2A (LGMD2A) [116,117]. Polymorphism within the calpain 10 gene (CAPN10) is associated with type 2 diabetes mellitus [118,119]. Overexpression of calpain 6 has been shown in uterine cervical cancer [120], while downregulation of calpain 9 is correlated with gastric cancer [9]. Inappropriate activation of calpain contributes to neurodegenerative diseases such as cerebral ischemia, multiple sclerosis, cataracts as well as Alzheimer's, Parkinson's, and Huntington's diseases [121]. Therefore, manipulating the proteolytic enzyme activity of calpain could provide useful therapies for the management of calpain-related diseases such as cancer, pathological angiogenesis, pulmonary hypertension, primary hypertension, muscular dystrophy, diabetes, and neurodegenerative disorders.