calpain and Cystic-Fibrosis

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (

Topics: Animals; Calpain; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Diaphragm; Humans; Inflammation; Lipopolysaccharides; Mice; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Ubiquitins

Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion.. PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n=48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays.. Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion.. Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.

Topics: Adult; Alleles; Animals; Calpain; Caveolin 1; Cell Adhesion; Cell Membrane; Cholesterol; Chronic Disease; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease Models, Animal; Disease Susceptibility; Endoplasmic Reticulum Stress; Female; Genotype; HL-60 Cells; Humans; Inflammation; Inflammation Mediators; Male; Membrane Microdomains; Mice; Mice, Knockout; Mutation; Neutrophils; Proteome; Proteomics; Respiratory Function Tests

Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion.

Topics: Adolescent; Adult; Aged; Calcium; Calpain; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Deletion; Gene Expression Regulation, Enzymologic; Homeostasis; Homozygote; Humans; Leukocytes, Mononuclear; Matrix Metalloproteinase 9; Middle Aged; Phosphorylation; Predictive Value of Tests; Protein Kinase C-alpha; Young Adult

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F(508)del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F(508)del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F(508)del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Calpain; Carrier Proteins; Cell Membrane; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Glycoproteins; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Oligopeptides; Phosphoproteins; Protein Binding; Sodium-Hydrogen Exchangers; Young Adult

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.

Topics: Animals; Calpain; Cell Membrane; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; HSP90 Heat-Shock Proteins; Humans; Immunoblotting; Immunoprecipitation; Molecular Chaperones; Mutation; Protein Transport; Rats; Rats, Inbred F344; Sequence Deletion

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

Topics: Adolescent; Adult; Calcium-Binding Proteins; Calpain; Case-Control Studies; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeletal Proteins; Enzyme Activation; Enzyme Assays; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Mutation, Missense; Phosphoproteins; Protein Isoforms; Protein Transport; Proteolysis; Sodium-Hydrogen Exchangers; Young Adult

Diabetes mellitus (DM) and abnormal glucose tolerance (IGT) are common in cystic fibrosis (CF). The loss of pancreatic beta-cells due to pancreatic fibrosis is thought to be one of the principal causes of diabetes in CF, but the aetiology of DM remains somewhat puzzling. Genetic factors may contribute to the development of CF related diabetes (CFRD). The purpose of this study was to investigate the role of polymorphisms in six genes on IGT or DM incidence. PCR and dHPLC were used to screen DNA samples for polymorphisms. Using 2-h oral glucose tolerance tests, 163 adult pancreatic insufficient CF patients have been subdivided in 3 groups: 54 NGT (normal glucose tolerance), 33 IGT and 76 CFRD. We found the first evidence for the association between CFRD and UCSNP-19 polymorphism in the CAPN10 gene. The UCSNP-19 genotype distribution differed significantly between NGT, IGT and CFRD groups. The difference reflected an increase in the 22 genotype (3 copies of 32-bp sequence) in IGT and CFRD patients (p=0.05). Odds ratio for the homozygote 22 versus homozygote 11 was 3.4 (p=0.02). All allele and genotype distributions for the other polymorphisms were similar in the three groups. In conclusion, our observations suggest that UCSNP-19 of CAPN10 may be involved in the pathogenesis of diabetes in CF.

Topics: Adult; Alleles; Blood Glucose; Calpain; Chromatography, High Pressure Liquid; Cystic Fibrosis; Diabetes Mellitus; DNA; Female; Genotype; Humans; Male; Polymerase Chain Reaction; Polymorphism, Genetic

We outline a general coalescent framework for using genotype data in linkage disequilibrium-based mapping studies. Our approach unifies two main goals of gene mapping that have generally been treated separately in the past: detecting association (i.e., significance testing) and estimating the location of the causative variation. To tackle the problem, we separate the inference into two stages. First, we use Markov chain Monte Carlo to sample from the posterior distribution of coalescent genealogies of all the sampled chromosomes without regard to phenotype. Then, averaging across genealogies, we estimate the likelihood of the phenotype data under various models for mutation and penetrance at an unobserved disease locus. The essential signal that these models look for is that in the presence of disease susceptibility variants in a region, there is nonrandom clustering of the chromosomes on the tree according to phenotype. The extent of nonrandom clustering is captured by the likelihood and can be used to construct significance tests or Bayesian posterior distributions for location. A novelty of our framework is that it can naturally accommodate quantitative data. We describe applications of the method to simulated data and to data from a Mendelian locus (CFTR, responsible for cystic fibrosis) and from a proposed complex trait locus (calpain-10, implicated in type 2 diabetes).

Topics: Alleles; Bayes Theorem; Calpain; Chromosome Mapping; Computer Simulation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diabetes Mellitus, Type 2; Disease Susceptibility; Genetic Variation; Haplotypes; Humans; Likelihood Functions; Linkage Disequilibrium; Markov Chains; Models, Genetic; Monte Carlo Method; Mutation; Pedigree; Penetrance; Polymorphism, Single Nucleotide; Quantitative Trait, Heritable; Recombination, Genetic

In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; Calcium-Binding Proteins; Calpain; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cystic Fibrosis; Cytochromes c; Densitometry; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Mitochondrial Proteins; Models, Biological; Models, Molecular; Neutrophils; Precipitin Tests; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Subcellular Fractions; Time Factors