Hiroshi Ishiguro

Cilia are hair-like organelles that protrude from the cell surface. In mammals, tracheal multiciliated cells (MCCs) play an important role in elimination of hazardous microorganisms by driving a unidirectional mucus flow. Although uniform orientation of ciliary beating is critical for the unidirectional flow, it remains unknown how MCCs establish uniform orientations and maintain identities of hundreds of ciliary membranes. This study focuses on investigating the roles of Tubulin Polymerization Promoting Family Member 3 (Tppp3) in MCC function. We generated a Tppp3-deficient mouse ( Tppp3 ^{∆ex2-4/∆ex2-4} ; Tppp3 knockout (KO)) and found that the Tppp3 KO mouse exhibited cough and hyposmia phenotype. The loss of Tppp3 disrupted the apical microtubules (MTs) meshwork in the tracheal MCCs, leading to random orientation and alignment of basal bodies (BBs) of the motile cilia. Unexpectedly, aberrant ciliary membrane fusions occurred in the trachea of the Tppp3 KO mice. We examined the underlying molecular mechanism of the ciliary membrane fusion by isolating the tracheal cilium. Liquid Chromatography-Mass Spectrometry (LC–MS) analysis as well as pharmacological analysis revealed that hyperaccumulation of a long chain ceramide at the ciliary membrane caused the membrane fusion. In addition, sensory cilia formation was impaired in the olfactory sensory neuron of the Tppp3 KO mice. Due to the lack of Tppp3, dendritic MT assembly that underlies long-range migration of BBs toward the cell surface was impaired. These findings demonstrate that Tppp3, as well as the defined intracellular MT architecture, regulate proper orientation/subcellular positioning of BBs and the independency of individual motile cilium membranes.

Cystic fibrosis (CF) is an autosomal recessive disease caused by pathogenic variants in CF transmembrane conductance regulator (CFTR). While CF is the most common hereditary disease in Caucasians, it is rare in East Asia. In the present study, we have examined clinical features and the spectrum of CFTR variants of CF patients in Japan. Clinical data of 132 CF patients were obtained from the national epidemiological survey since 1994 and CF registry. From 2007 to 2022, 46 patients with definite CF were analyzed for CFTR variants. All exons, their boundaries, and part of promoter region of CFTR were sequenced and the presence of large deletion and duplications were examined by multiplex ligation-dependent probe amplification. CF patients in Japan were found to have chronic sinopulmonary disease (85.6%), exocrine pancreatic insufficiency (66.7%), meconium ileus (35.6%), electrolyte imbalance (21.2%), CF-associated liver disease (14.4%), and CF-related diabetes (6.1%). The median survival age was 25.0 years. The mean BMI percentile was 30.3%ile in definite CF patients aged < 18 years whose CFTR genotypes were known. In 70 CF alleles of East Asia/Japan origin, CFTR-dele16-17a-17b was detected in 24 alleles, the other variants were novel or very rare, and no pathogenic variants were detected in 8 alleles. In 22 CF alleles of Europe origin, F508del was detected in 11 alleles. In summary, clinical phenotype of Japanese CF patients is similar to European patients, but the prognosis is worse. The spectrum of CFTR variants in Japanese CF alleles is entirely different from that in European CF alleles.

TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting both upper and lower motor neurons. Besides motor symptoms, patients with ALS often develop nonneuronal signs including glucose intolerance, but the underlying pathomechanism is still controversial, i.e., whether it is impaired insulin secretion and/or insulin resistance. Here, we showed that ALS subjects reduced early-phase insulin secretion and that the nuclear localization of TDP-43 was lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibited exocytosis by downregulating CaV1.2 calcium channels, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6) and β cell–specific Tardbp–knockout mice. Overexpression of CaV1.2 restored early-phase insulin secretion in Tardbp–knocked-down MIN6 cells. Our findings suggest that TDP-43 regulates cellular exocytosis mediated by L-type voltage–dependent calcium channels and, thus, plays an important role in the early phase of insulin secretion by pancreatic islets. Thus, nuclear loss of TDP-43 is implicated in not only the selective loss of motor neurons, but also in glucose intolerance due to impaired insulin secretion at an early stage of ALS.

Caffeine is contained in coffee, tea, and numerous beverages and foods. We examined the direct effects of caffeine on the physiological function of pancreatic duct cells by using interlobular duct segments isolated from guinea pig pancreas. The rate of fluid secretion was continuously measured by monitoring the luminal volume of isolated duct segments. Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by microfluorometry in ducts loaded with Fura-2. Both secretin-stimulated and acetylcholine (ACh)-stimulated fluid secretions were substantially and reversibly inhibited by relatively low concentrations of caffeine as low as 0.03 mM relevant to blood levels after ingestion of caffeine-containing beverages. Caffeine inhibited ACh-induced elevation of [Ca2+]i and secretin-induced fluctuation of [Ca2+]i. Caffeine abolished thapsigargin-induced intracellular Ca2+ release but did not affect the entry of extracellular Ca2+. Caffeine (0.05 mM) abolished ethanol (1 mM)-induced fluid hypersecretion in secretin-stimulated pancreatic duct. Low concentrations of caffeine directly inhibit pancreatic ductal fluid secretion stimulated by secretin or ACh and also ethanol-induced fluid hypersecretion. The inhibition by caffeine seems to be mediated by the blockade of intracellular Ca2+ mobilization. Daily intake of caffeine may reduce the volume of pancreatic juice secretion.

Key points: The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO₃⁻ concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea‐pig pancreatic ducts. The model was readily able to secrete 140 mm HCO₃⁻. Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO₃⁻ concentrations is to minimize the secretion of Cl⁻ ions. These findings help to clarify the mechanism responsible for pancreatic HCO₃⁻ secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. A computational model of guinea‐pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO₃⁻ ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least‐squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP‐stimulated secretion were well replicated by increasing the activities of the basolateral Na⁺‐HCO₃⁻ cotransporter (NBC1) and apical Cl⁻/HCO₃⁻ exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K⁺ permeability and apical Cl⁻ and HCO₃⁻ permeabilities (CFTR), and reducing the activity of the basolateral Cl⁻/HCO₃⁻ exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO₃⁻ at a rate of ∼3 nl min⁻¹ mm⁻², which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl⁻/HCO₃⁻ exchange via SLC26A6 at the apical membrane were able to support a HCO₃⁻‐rich secretion. Raising the HCO₃⁻/Cl⁻ permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO₃⁻ concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl⁻ concentration following cAMP stimulation and thereby maximizing the secreted HCO₃⁻ concentration. The addition of a basolateral Na⁺‐K⁺‐2Cl⁻ cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl⁻ and resulted in a lower secreted HCO₃⁻ concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl⁻ secretion is the main requirement for secreting 140 mm HCO₃⁻.

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl⁻ has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO₃⁻) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl⁻/HCO₃⁻ exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ∼60%, impaired CFTR-mediated HCO₃⁻/Cl⁻ transport activity to 50–60%, and impaired CFTR-coupled Cl⁻/HCO₃⁻ exchange activity to 20–30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.