Increased cytosolic calcium in cystic fibrosis neutrophils effect on stimulus-secretion coupling

A disorder of calcium homeostasis has been related to the pathogenesis of Cystic Fibrosis (CF). The Authors have studied the relationship between the cytosolic free calcium concentration ([Ca2+]i), the amount of Ca2+ released from endogenous stores and the secretory response in CF neutrophils. Significantly elevated resting [Ca2+]i and depressed Ca2+ release induced by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is present in CF neutrophils. In the absence of exogenous Ca2+ the secretory response of CF neutrophils after a weak stimulus such as Cytochalasin B (CB) is greater than in normal neutrophils, while a depressed secretion of azurophilic granules is evident in CF neutrophils stimulated by CB + FMLP. The data confirm the hypothesis of an altered Ca2+ homeostasis in CF cells. Cystic Fibrosis (CF), an autosomal recessive exocrinopathy, is characterized by secretory abnormalities and ion transport dysfunctions (for review see 1,2). Since intracellular Ca2+ seems to play a role in stimulus-secretion coupling and ion movements, several aspects of Ca2+ homeostasis have been investigated in CF. The total Ca2+ content has been reported to be increased in fibroblast cultures and in lymphocytes (3,4,5) and mitochondrial Ca2+ uptake was found elevated in fibroblast cultures (6). An elevated free cytosolic calcium concentration ([Ca2+]i) has been recently reported in buccal epithelial cells (7), while normal concentration has been found in lymphocytes and Epstein Barr virus transformed lymphoblasts (5,8). The present paper shows the results of a study in human neutrophils, a cell whose several functions such as secretion, movement and respiratory burst are in some way regulated by Ca2+. The data report that in neutrophils of CF patients the resting [Ca2+]i is higher and the secretory response is partly modified.     

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca²⁺) homeostasis. In this study, we discovered that the Ca²⁺ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca²⁺ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca²⁺ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.     

Absent secretion to vasoactive intestinal peptide in cystic fibrosis airway glands

We are testing the hypothesis that the malfunctioning of airway gland serous cells is a component of cystic fibrosis (CF) airway disease. CF is caused by mutations that disrupt CF transmembrane conductance regulator, an anion channel essential for proper fluid secretion in some epithelia. Submucosal glands supply most of the mucus in upper airways, and gland serous cells are the primary site of CF transmembrane conductance regulator expression in airways. We have discovered a major defect in CF glands by in situ optical monitoring of secretions from single human airway glands. CF glands did not secrete to agents that elevated [cAMP](i) (0 responses/450 glands, 8 subjects), whereas glands were responsive in all donor tracheas (605/827 glands, 15 subjects) and in bronchi from subjects who were transplanted because of other lung diseases (148/166 glands, n = 10). CF glands secreted to cholinergic stimulation, and serous cells were abundant in glands from all CF subjects. The complete absence of secretion to agents that elevate [cAMP](i) suggests that altered secretion of gland mucus could contribute to CF lung disease.     

CFTR: folding, misfolding and correcting the ΔF508 conformational defect

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.     

Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.     

Cell membrane permeability in hypertension

A great deal of evidence has been presented which suggests that a generalized increase in cell membrane permeability to various ions is a fundamental defect associated with hypertension. In addition, some of the evidence is supportive of a central role of this defect in the pathogenesis of hypertension. The source of this defect may lie in the reduced calcium binding capacity of the cell membrane. Because of this reduced capacity the membrane is less stable and is more permeable to various ions.     

Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair

Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect.     

Antioxidants in cystic fibrosis. Conclusions from the CF antioxidant workshop, Bethesda, Maryland, November 11-12, 2003

Although great strides are being made in the care of individuals with cystic fibrosis (CF), this condition remains the most common fatal hereditary disease in North America. Numerous links exist between progression of CF lung disease and oxidative stress. The defect in CF is the loss of function of the transmembrane conductance regulator (CFTR) protein; recent evidence that CFTR expression and function are modulated by oxidative stress suggests that the loss may result in a poor adaptive response to oxidants. Pancreatic insufficiency in CF also increases susceptibility to deficiencies in lipophilic antioxidants. Finally the airway infection and inflammatory processes in the CF lung are potential sources of oxidants that can affect normal airway physiology and contribute to the mechanisms causing characteristic changes associated with bronchiectasis and loss of lung function. These multiple abnormalities in the oxidant/antioxidant balance raise several possibilities for therapeutic interventions that must be carefully assessed.     

Ultrastructural and immunohistochemical characterization of submandibular duct cells in culture and modification of outgrowth differentiation by manipulation of calcium ion concentration

Summary The recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation. This work was supported by Public Health Service grant AM 11028, Department of Health and Human Services, Washington, DC.     

Osteopetrorickets due to Snx10 Deficiency in Mice Results from Both Failed Osteoclast Activity and Loss of Gastric Acid-Dependent Calcium Absorption

Mutations in sorting nexin 10 (Snx10) have recently been found to account for roughly 4% of all human malignant osteopetrosis, some of them fatal. To study the disease pathogenesis, we investigated the expression of Snx10 and created mouse models in which Snx10 was knocked down globally or knocked out in osteoclasts. Endocytosis is severely defective in Snx10-deficent osteoclasts, as is extracellular acidification, ruffled border formation, and bone resorption. We also discovered that Snx10 is highly expressed in stomach epithelium, with mutations leading to high stomach pH and low calcium solubilization. Global Snx10-deficiency in mice results in a combined phenotype: osteopetrosis (due to osteoclast defect) and rickets (due to high stomach pH and low calcium availability, resulting in impaired bone mineralization). Osteopetrorickets, the paradoxical association of insufficient mineralization in the context of a positive total body calcium balance, is thought to occur due to the inability of the osteoclasts to maintain normal calcium–phosphorus homeostasis. However, osteoclast-specific Snx10 knockout had no effect on calcium balance, and therefore led to severe osteopetrosis without rickets. Moreover, supplementation with calcium gluconate rescued mice from the rachitic phenotype and dramatically extended life span in global Snx10-deficient mice, suggesting that this may be a life-saving component of the clinical approach to Snx10-dependent human osteopetrosis that has previously gone unrecognized. We conclude that tissue-specific effects of Snx10 mutation need to be considered in clinical approaches to this disease entity. Reliance solely on hematopoietic stem cell transplantation can leave hypocalcemia uncorrected with sometimes fatal consequences. These studies established an essential role for Snx10 in bone homeostasis and underscore the importance of gastric acidification in calcium uptake.     

Calcium signaling is involved in dynein-dependent microtubule organization

The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Deltaeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca(2+)/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Deltaeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.     

Cystic fibrosis affects chloride and sodium channels in human airway epithelia

Abnormalities of epithelial function in cystic fibrosis (CF) have been linked to defects in cell membrane permeability to chloride or sodium ions. Recently, a class of chloride channels in airway epithelial cells have been reported to lack their usual sensitivity to phosphorylation via cAMP-dependent protein kinase, suggesting that CF could be due to a single genetic defect in these channels. We have examined single chloride and sodium channels in control and CF human nasal epithelia using the patch-clamp technique. The most common chloride channel was not the one previously associated with CF, but it was also abnormal in CF cells. In addition, the number of sodium channels was unusually high in CF. These findings suggest a wider disturbance of ion channel properties in CF than would be produced by a defect in a single type of channel.     

Can correcting the ΔF508-CFTR proteostasis-defect rescue CF lung disease?

Protein homeostasis (proteostasis) generates and maintains individual proteins in their folded and functional-competent states. The components of the cellular proteostasis machinery also dictate the functional lifetime of a protein by constantly regulating its conformation, concentration and subcellular location. The autosomal recessive disease cystic fibrosis (CF) is caused by a proteostasis-defect in CF transmembrane conductance regulator (CFTR). The most common CF mutation leading to this proteostasis-defect is the deletion of a phenylalanine residue at position 508 (ΔF508) of the CFTR protein. This ΔF508-CFTR protein is prone to aberrant folding, increased ER-associated degradation, atypical intracellular trafficking and reduced stability at the apical membrane. This ΔF508-CF proteostasis-defect leads to an obstructive lung disease characterized by impaired ion transport in airway epithelial cells, mucus buildup in air space and chronic airway inflammation. We assess here whether correcting the underlying defect in ΔF508-CFTR protein processing using therapeutic proteostasis regulators can treat chronic CF lung disease. As a proof of concept, recent studies support that the selective modulation of mutant-CFTR proteostasis may offer promising therapies to reverse chronic CF lung disease.     

Endoplasmic reticulum calcium transport ATPase expression during differentiation of colon cancer and leukaemia cells

The calcium homeostasis of the endoplasmic reticulum (ER) is connected to a multitude of cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Calcium is accumulated in the ER by various biochemically distinct sarco/endoplasmic reticulum calcium transport ATPase isoenzymes (SERCA isoforms). Experimental data indicate that the SERCA composition of some carcinoma and leukaemia cell types undergoes significant changes during differentiation, and that this is accompanied by modifications of SERCA-dependent calcium accumulation in the ER. Because ER calcium homeostasis can also influence cell differentiation, we propose that the modulation of the expression of various SERCA isoforms, and in particular, the induction of the expression of SERCA3-type proteins, is an integral part of the differentiation program of some cancer and leukaemia cell types. The SERCA content of the ER may constitute a new parameter by which the calcium homeostatic characteristics of the organelle are adjusted. The cross-talk between ER calcium homeostasis and cell differentiation may have some implications for the better understanding of the signalling defects involved in the acquisition and maintenance of the malignant phenotype.     

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.     

Intracellular calcium in cystic fibrosis heterozygotes

Increased intracellular calcium (Ca) has been reported in several cell types in cystic fibrosis (CF). Because CF is an autosomal recessive trait examination of asymptomatic obligate carriers (HZ) of the gene is a powerful way to determine the relevance of this observation to the abnormal gene product. We report here that Ca as determined by atomic absorption spectrophotometry in cultured skin fibroblasts and circulating lymphocytes is greater in HZ than in control cells. Since an intracellular Ca increase is expressed in HZ the Ca differences in CF likely reflect action of the gene product responsible for CF and not some secondary or tertiary effect of the disease.     

Alterations in calcium homeostasis as biological marker for mild Alzheimer's disease?

The calcium hypothesis of neurodegenerative disorders such as Alzheimer's disease (AD) suggests that altered cytosolic Ca(2+) levels ( Ca(2+) (i)) and/or disturbances in Ca2+ homeostasis concern cellular mechanisms underlying neuronal pathology. To search for a diagnostic marker of Alzheimer's disease, we measured cytosolic calcium concentrations in platelets of AD patients, age-matched control subjects (AMC), and vascular dementia (VD) patients. The ( Ca(2+) (i)) was determined using long wavelength indicator Fluo-3AM in 21 mild AD patients, 17 AMC, and 23 patients with VD. The basal values of [Ca(2+)](i) were significantly lower in AD compared to AMC. After the addition of 1 mM calcium, the [Ca(2+)](i) markedly increased in platelets of AD compared to AMC and VD. Measurement of calcium homeostasis could provide a very sensitive, but less specific biological marker of AD. These results support the hypothesis that influencing calcium homeostasis may provide a therapeutic strategy in dementia.     

Impaired regulatory volume decrease in freshly isolated cholangiocytes from cystic fibrosis mice: implications for cystic fibrosis transmembrane conductance regulator effect on potassium conductance

Various K(+) and Cl(-) channels are important in cell volume regulation and biliary secretion, but the specific role of cystic fibrosis transmembrane conductance regulator in cholangiocyte cell volume regulation is not known. The goal of this research was to study regulatory volume decrease (RVD) in bile duct cell clusters (BDCCs) from normal and cystic fibrosis (CF) mouse livers. Mouse BDCCs without an enclosed lumen were prepared as described (Cho, W. K. (2002) Am. J. Physiol. 283, G1320-G1327). The isotonic solution consisted of HEPES buffer with 40% of the NaCl replaced with isomolar amounts of sucrose, whereas hypotonic solution was the same as isotonic solution without sucrose. The cell volume changes were indirectly assessed by measuring cross-sectional area (CSA) changes of the BDCCs using quantitative videomicroscopy. Exposure to hypotonic solutions increased relative CSAs of normal BDCCs to 1.20 +/- 0.01 (mean +/- S.E., n = 50) in 10 min, followed by RVD to 1.07 +/- 0.01 by 40 min. Hypotonic challenge in CF mouse BDCCs also increased relative CSA to 1.20 +/- 0.01 (n = 53) in 10 min but without significant recovery. Coadministration of the K(+)-selective ionophore valinomycin restored RVD in CF mouse BDCCs, suggesting that the impaired RVD was likely from a defect in K(+) conductance. Moreover, this valinomycin-induced RVD in CF mice was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, indicating that it is not from nonspecific effects. Neither cAMP nor calcium agonists could reverse the impaired RVD seen in CF cholangiocytes. Our conclusion is that CF mouse cholangiocytes have defective RVD from an impaired K(+) efflux pathway, which could not be reversed by cAMP nor calcium agonists.     

Sulfate transport in normal and cystic fibrosis fibroblasts.

The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts.     

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.