Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium dependence of the founder members of the family, the ubiquitously expressed enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain family includes more than a dozen members with sequence relatedness to the catalytic subunits of calpain 1 and 2. Calpains have a modular domain architecture. By convention, the family is subdivided into classical and nonclassical calpains, according to the presence or absence, respectively, of a calcium-binding penta-EF-hand module in domain IV of the protein (1). Classical calpains include calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7, 10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an essential role for calpains during embryonic development. Knock-out of the small regulatory subunit (Capn4) results in embryonic lethality (2, 3). Similarly, inactivation of the Capn2 gene blocks development between the morula and blastocyst stage (4). In humans, mutations in CAPN3 underlie limb-girdle muscular dystrophy-2A, and polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus (5, 6).Even under conditions of calcium overload, where calpains are presumably activated maximally, only a subset (<5%) of cellular proteins are hydrolyzed (7). Calpains typically cleave their substrates at a limited number of sites to generate large polypeptide fragments that, in many cases, retain bioactivity. Thus, under physiological conditions, calpains probably participate in the regulation of protein function rather than in non-specific protein degradation.More than 100 proteins have been shown to serve as calpain substrates in vitro, including cytoskeletal proteins (8), signal transduction molecules (9), ion channels (10), and receptors (11). In vivo, calpains are believed to function in myoblast fusion (12), long term potentiation (13), and cellular mobility (14). Unregulated calpain activity, secondary to intracellular calcium overload, is associated with several pathological conditions, including Alzheimer disease (15), animal models of cataract (16), myocardial (17), and cerebral ischemia (18).In addition to their domain structure, calpains are often classified according to their tissue expression patterns. Calpain 1, 2, and 10 are widely expressed in mammalian tissues, but other members of the calpain family show tissue-specific expression patterns. Calpain 8, for example, is a stomach-specific calpain (19), whereas expression of calpain 9 is restricted to tissues of the digestive tract (20). The expression of calpain 3 was originally thought to be limited to skeletal muscle (21), but splice variants of calpain 3 have since been detected in a range of tissues. At least 12 isoforms of calpain 3 have been described in rodents (22), of which several are expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88 (retina) (23).Calpains have been studied intensively in the ocular lens because of their suspected involvement in lens opacification (cataract). Calpain-mediated proteolysis of lens crystallin proteins causes increased light scatter (24). Unregulated activation of calpains is observed in rodent cataract models (25), where calpain-mediated degradation of crystallin proteins (26) and cytoskeletal elements (27) is commonly observed. Calpain inhibitors are effective in delaying or preventing cataract in vitro (28, 29) and in vivo (30, 31).It is likely, however, that calpains have important physiological roles in the lens beyond their involvement in tissue pathology. Terminal differentiation of lens fiber cells involves a series of profound morphological and biochemical transformations. For example, differentiating lens fiber cells undergo an enormous (>100-fold) increase in cell length, accompanied by extensive remodeling of the plasma membrane system (32). Early in the differentiation process, fusion pores are established between cells, as neighboring fibers are incorporated into the lens syncytium (33). A later stage of fiber cell differentiation involves the dissolution of all intracellular organelles, a process that is thought to eliminate light-scattering particles from the light path and contribute to the transparency of the tissue (34). Any or all of these phenomena might require the developmentally regulated activation of calpains. This is consistent with our previous observation that in calpain 3 knock-out mice the transition zone is altered, suggesting a change in the differentiation program (35).In the current study, therefore, we examined the depth-dependent expression pattern and activity of calpains in the mouse lens. Fluorogenic substrates were microinjected into the intact lens to visualize calpain activity directly, and proteomic approaches were used to identify endogenous calpain substrates. The cleavage pattern of one of these, αII-spectrin, was examined in detail. Immunocytochemical and immunoblot analysis with wild type and calpain 3-null lenses indicated that αII-spectrin is a specific calpain 3 substrate in maturing lens fiber cells. Together, the data suggest that calpains are activated relatively late in fiber cell differentiation and may contribute to the remodeling of the membrane cytoskeleton that accompanies fiber cell maturation.     

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Characterization of Enantiomeric Bile Acid-induced Apoptosis in Colon Cancer Cell Lines

Bile acids are steroid detergents that are toxic to mammalian cells at high concentrations; increased exposure to these steroids is pertinent in the pathogenesis of cholestatic disease and colon cancer. Understanding the mechanisms of bile acid toxicity and apoptosis, which could include nonspecific detergent effects and/or specific receptor activation, has potential therapeutic significance. In this report we investigate the ability of synthetic enantiomers of lithocholic acid (ent-LCA), chenodeoxycholic acid (ent-CDCA), and deoxycholic acid (ent-DCA) to induce toxicity and apoptosis in HT-29 and HCT-116 cells. Natural bile acids were found to induce more apoptotic nuclear morphology, cause increased cellular detachment, and lead to greater capase-3 and -9 cleavage compared with enantiomeric bile acids in both cell lines. In contrast, natural and enantiomeric bile acids showed similar effects on cellular proliferation. These data show that bile acid-induced apoptosis in HT-29 and HCT-116 cells is enantiospecific, hence correlated with the absolute configuration of the bile steroid rather than its detergent properties. The mechanism of LCA- and ent-LCA-induced apoptosis was also investigated in HT-29 and HCT-116 cells. These bile acids differentially activate initiator caspases-2 and -8 and induce cleavage of full-length Bid. LCA and ent-LCA mediated apoptosis was inhibited by both pan-caspase and selective caspase-8 inhibitors, whereas a selective caspase-2 inhibitor provided no protection. LCA also induced increased CD95 localization to the plasma membrane and generated increased reactive oxygen species compared with ent-LCA. This suggests that LCA/ent-LCA induce apoptosis enantioselectively through CD95 activation, likely because of increased reactive oxygen species generation, with resulting procaspase-8 cleavage.Bile acids are physiologic steroids that are necessary for the proper absorption of fats and fat-soluble vitamins. Their ability to aid in these processes is largely due to their amphipathic nature and thus their ability to act as detergents. Despite the beneficial effects, high concentrations of bile acids are toxic to cells (1-11). High fat western diets induce extensive recirculation of the bile acid pool, resulting in increased exposure of the colonic epithelial cells to these toxic steroids (12, 13). A high fat diet is also a risk factor for colon carcinogenesis; increased bile acid exposure is responsible for some of this risk. Bile acids can contribute to both colon cancer formation and progression, and their effects on colonic proliferation and apoptosis aid this process by disrupting the balance between cell growth and cell death, as well as helping to select for bile acid-resistant cells (14, 15).In colonocyte-derived cell lines bile acid-induced apoptosis is thought to proceed through mitochondrial destabilization with resulting mitochondrial permeability transition formation and cytochrome c release as well as generation of oxidative stress (1, 9-11). Bile acid-induced apoptosis has also been extensively explored in hepatocyte derived cell lines with mechanisms including mitochondria dysfunction (16-23), endoplasmic reticulum stress (24), ligand-independent activation of death receptor pathways (18, 25-28), and modulation of cellular apoptotic and anti-apoptotic Bcl-2 family proteins (29).Although ample evidence exists for multiple mechanisms of bile acid-induced apoptosis, the precise interactions responsible for initiating these apoptotic pathways are still unclear. Bile acids have been shown to interact directly with specific receptors (30, 31). These steroids can also initiate cellular signaling through nonspecific membrane perturbations (32), and evidence exists showing that other simple detergents (i.e. Triton X-100) are capable of inducing caspase cleavage nonspecifically with resultant apoptosis (33). Therefore, hydrophobic bile acids may interact nonspecifically with cell membranes to alter their physical properties, bind to receptors specific for these steroids, or utilize a combination of both specific and nonspecific interactions to induce apoptosis.Bile acid enantiomers could be useful tools for elucidating mechanisms of bile acid toxicity and apoptosis. These enantiomers, known as ent-bile acids, are synthetic nonsuperimposable mirror images of natural bile acids with identical physical properties except for optical rotation. Because bile acids are only made in one absolute configuration naturally, ent-bile acids must be constructed using a total synthetic approach. Recently we reported the first synthesis of three enantiomeric bile acids: ent-lithocholic acid (ent-LCA),² ent-chenodeoxycholic acid (ent-CDCA), and ent-deoxycholic acid (ent-DCA) (Fig. 1) (34, 35). Enantiomeric bile acids have unique farnesoid X receptor, vitamin D receptor, pregnane X receptor, and TGR5 receptor activation profiles compared with the corresponding natural bile acids (34). This illustrates that natural and enantiomeric bile acids interact differently within chiral environments because of their distinct three-dimensional configurations (Fig. 1). Despite these differences in chiral interactions, ent-bile acids have physical properties identical to those of their natural counterparts including solubility and critical micelle concentrations (34, 35). With different receptor interaction profiles and identical physical properties compared with natural bile acids, ent-bile acids are ideal compounds to differentiate between the receptor-mediated and the non-receptor-mediated functions of natural bile acids.Open in a separate windowFIGURE 1.Natural and enantiomeric bile acids. Structures and three-dimensional projection views of natural LCA, CDCA, DCA, and their enantiomers (ent-LCA, ent-CDCA, and ent-DCA). The three-dimensional ent-steroid structure is rotated 180° around the long axis for easier comparison with the natural steroid.In this study we explore the enantioselectivity of LCA-, CDCA-, and DCA-mediated toxicity and apoptosis in two human colon adenocarcinoma cell lines, HT-29 and HCT-116. Because the mechanism of natural LCA induced apoptosis has never been characterized, we then examined in more detail LCA- and ent-LCA-mediated apoptosis in colon cancer cells. These studies will not only explore the LCA apoptotic mechanism but will also determine whether ent-LCA signals through similar cellular pathways.     

Sleeping Beauty Mouse Models Identify Candidate Genes Involved in Gliomagenesis

Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.     

Piwi Genes Are Dispensable for Normal Hematopoiesis in Mice

Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized “piwi” refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal lineage reconstitution after competitive transplantation. We further show that the three piwi genes are dispensable during hematopoietic recovery after myeloablative stress by 5-FU. Collectively, our data suggest that the function of the piwi gene subfamily is not required for normal adult hematopoiesis.     

ATG18 and FAB1 Are Involved in Dehydration Stress Tolerance in Saccharomyces cerevisiae

Recently, different dehydration-based technologies have been evaluated for the purpose of cell and tissue preservation. Although some early results have been promising, they have not satisfied the requirements for large-scale applications. The long experience of using quantitative trait loci (QTLs) with the yeast Saccharomyces cerevisiae has proven to be a good model organism for studying the link between complex phenotypes and DNA variations. Here, we use QTL analysis as a tool for identifying the specific yeast traits involved in dehydration stress tolerance. Three hybrids obtained from stable haploids and sequenced in the Saccharomyces Genome Resequencing Project showed intermediate dehydration tolerance in most cases. The dehydration resistance trait of 96 segregants from each hybrid was quantified. A smooth, continuous distribution of the anhydrobiosis tolerance trait was found, suggesting that this trait is determined by multiple QTLs. Therefore, we carried out a QTL analysis to identify the determinants of this dehydration tolerance trait at the genomic level. Among the genes identified after reciprocal hemizygosity assays, RSM22, ATG18 and DBR1 had not been referenced in previous studies. We report new phenotypes for these genes using a previously validated test. Finally, our data illustrates the power of this approach in the investigation of the complex cell dehydration phenotype.     

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca²⁺ channels. Inhibition of the oxidative response impaired Cl⁻ efflux, K⁺ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.     

Autoradiography of Developing Syncytia in Cotton Roots Infected with Meloidogyne incognita

Cotton (Gossypium hirsutum) seedlings, uniformly infected with Meloidogyne incognita, were exposed for periods of 1-15 days to a nutrient solution containing tritium-labelled thymidine. Syncytium formation began with the amalgamation of cells near the nematode head, and was followed by synchronized mitoses of the nuclei which had been incorporated into a single cell. Syncytial nuclei synthesized DNA in roots harvested 3, 6, 9, 12, and 15 days after inoculation. Seedlings transferred from unlabelled to labelled nutrient solution 9 days after inoculation, and grown for 6 more days, contained some syncytial nuclei which did not become labelled. Giant-cell nuclei increased in size and, in many cases, all nuclei in one giant cell of a set showed active DNA synthesis at about the time the nematode molted to the adult stage.