Peter A. Greer

Disruption of the Murine Calpain Small Subunit Gene, Capn4: Calpain Is Essential for Embryonic Development but Not for Cell Growth and Division

ABSTRACT Calpains are a family of Ca2+-dependent intracellular cysteine proteases, including the ubiquitously expressed μ- and m-calpains. Both μ- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genesCapn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of μ- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both μ- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.

ABSTRACT Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N′-nitro-N′-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

Reduced Cell Migration and Disruption of the Actin Cytoskeleton in Calpain-deficient Embryonic Fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The μ- and m-calpains consist of a μ- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained fromCapn4−/− mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4−/− cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4−/− cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, α-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4+/+ andCapn4−/− cells. FAK, α-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in theCapn4−/− cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4−/− embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.

Closing in on the biological functions of fps/fes and fer

Fps/Fes and Fer are the only known members of a distinct subfamily of the non-receptor protein-tyrosine kinase family. Recent studies indicate that these kinases have roles in regulating cytoskeletal rearrangements and inside–out signalling that accompany receptor–ligand, cell–matrix and cell–cell interactions. Genetic analysis using transgenic mouse models also implicates these kinases in the regulation of inflammation and innate immunity.

Ubiquitous Calpains Promote Caspase-12 and JNK Activation during Endoplasmic Reticulum Stress-induced Apoptosis

Ubiquitously expressed μ- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of μ- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpains role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.

Continuous association of cadherin with β-catenin requires the non-receptor tyrosine-kinase Fer

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by α- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

Apicomplexan Parasites Co-Opt Host Calpains to Facilitate Their Escape from Infected Cells

Let Me Out Apicomplexan parasites like Plasmodium falciparum, which causes malaria, and Toxoplasma gondii, which cause toxoplasmosis, replicate inside animal host cells. In order for infections to spread successfully within the host from cell to cell, daughter parasites after replication need to be able to escape from their incubator cell. In the course of studies intended to elucidate the functions of proteases during parasite infection, Chandramohanadas et al. (p. 794, published online 2 April) noted that host cell calpain is the only protease present at the right time and place to facilitate the egress of malaria parasites from infected red blood cells. Parasite egress from infected resealed erythrocytes was prevented when calpain was removed. Moreover, T. gondii was unable to escape efficiently from murine fibroblast knockouts lacking a calpain regulatory subunit. A host protease helps newly replicated microbial parasites escape from incubator cells. Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either small interfering RNA–mediated suppression or genetic deletion of calpain activity and could be restored by genetic complementation.

m-Calpain is required for preimplantation embryonic development in mice

Backgroundμ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice.ResultsTo distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage.ConclusionWe conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Ubiquitous Calpains Promote Both Apoptosis and Survival Signals in Response to Different Cell Death Stimuli

The μ- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both μ- and m-calpain activities. Capn4–/– MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4–/– MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor α-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4–/– MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4–/– MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor α- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.

The Fps/Fes protein-tyrosine kinase promotes angiogenesis in transgenic mice

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase known to be highly expressed in hematopoietic cells. To investigate fps/fes biological function, an activating mutation was introduced into the human fps/fes gene which directs amino-terminal myristylation of the Fps/Fes protein. This mutant, myristylated protein induced transformation of Rat-2 fibroblasts. The mutant fps/fes allele was incorporated into the mouse germ line and was found to be appropriately expressed in transgenic mice, in a tissue-specific pattern indistinguishable from that of the endogenous mouse gene. These mice displayed widespread hypervascularity, progressing to multifocal hemangiomas. High levels of both the transgenic human and endogenous murine fps/fes transcripts were detected in vascular tumors by using RNase protection, and fps/fes transcripts were localized to endothelial cells of both the vascular tumors and normal blood vessels by in situ RNA hybridization. Primary human umbilical vein endothelial cultures were also shown to express fps/fes transcripts and the Fps/Fes tyrosine kinase. These results indicate that fps/fes expression is intrinsic to cells of the vascular endothelial lineage and suggest a direct role of the Fps/Fes protein-tyrosine kinase in the regulation of angiogenesis.