Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in c ongenital p olycystic k idney ( cpk ) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models ( cpk , rescued‐ cpk (r‐ cpk ), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type ( wt ), cpk ). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r ‐cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 ( PKHD1 ) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver ( cyli ), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd1 cyli/cyli ( cyli ) mice exhibit a severe liver pathology but lack renal disease. Further analysis revealed that several alternatively spliced Pkhd1 mRNA, all containing exon 48, were expressed in cyli kidneys, but in lower abundance than in wild-type kidneys, suggesting that these transcripts escaped from nonsense-mediated decay (NMD). We identified an AAAAAT motif in exon 48 upstream of the cyli mutation which could enable ribosomal frameshifting, thus potentially allowing production of sufficient amounts of FPC for renoprotection. This mechanism, expressed in a species-specific fashion, may help explain the disparities in the renal phenotype observed between Pkhd1 mutant mice and patients with PKHD1 -related disease. Key messages The Pkhd1 cyli/cyli mouse expresses cystic liver disease, but no kidney phenotype. Pkhd1 mRNA expression is decreased in cyli liver and kidneys compared to wild-type. Ribosomal frameshifting may be responsible for Pkhd1 mRNA escape from NMD. Pkhd1 mRNA escape from NMD could contribute to the absent kidney phenotype.

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is a severe, hereditary, hepato-renal fibrocystic disorder that leads to early childhood morbidity and mortality. Typical forms of ARPKD are caused by pathogenic variants in the PKHD1 gene, which encodes the fibrocystin/polyductin (FPC) protein. MYC overexpression has been proposed as a driver of renal cystogenesis, but little is known about MYC expression in recessive PKD. In the current study, we provide the first evidence that MYC is overexpressed in kidneys from ARPKD patients and confirm that MYC is upregulated in cystic kidneys from cpk mutant mice. In contrast, renal MYC expression levels were not altered in several Pkhd1 mutant mice that lack a significant cystic kidney phenotype. We leveraged previous observations that the carboxy-terminus of mouse FPC (FPC-CTD) is proteolytically cleaved through Notch-like processing, translocates to the nucleus, and binds to double stranded DNA, to examine whether the FPC-CTD plays a role in regulating MYC/Myc transcription. Using immunofluorescence, reporter gene assays, and ChIP, we demonstrate that both human and mouse FPC-CTD can localize to the nucleus, bind to the MYC/Myc P1 promoter, and activate MYC/Myc expression. Interestingly, we observed species-specific differences in FPC-CTD intracellular trafficking. Furthermore, our informatic analyses revealed limited sequence identity of FPC-CTD across vertebrate phyla and database queries identified temporal differences in PKHD1/Pkhd1 and CYS1/Cys1 expression patterns in mouse and human kidneys. Given that cystin, the Cys1 gene product, is a negative regulator of Myc transcription, these temporal differences in gene expression could contribute to the relative renoprotection from cystogenesis in Pkhd1-deficient mice. Taken together, our findings provide new mechanistic insights into differential mFPC-CTD and hFPC-CTD regulation of MYC expression in renal epithelial cells, which may illuminate the basis for the phenotypic disparities between human patients with PKHD1 pathogenic variants and Pkhd1-mutant mice.

Autosomal recessive polycystic kidney disease (ARPKD) is caused primarily by mutations in PKHD1, encoding fibrocystin (FPC), but Pkhd1 mutant mice failed to reproduce the human phenotype. In contrast, the renal lesion in congenital polycystic kidney (cpk) mice, with a mutation in Cys1 and cystin protein loss, closely phenocopies ARPKD. Although the nonhomologous mutation diminished the translational relevance of the cpk model, recent identification of patients with CYS1 mutations and ARPKD prompted the investigations described herein. We examined cystin and FPC expression in mouse models (cpk, rescued-cpk (r-cpk), Pkhd1 mutants) and mouse cortical collecting duct (CCD) cell lines (wild type (wt), cpk). We found that cystin deficiency caused FPC loss in both cpk kidneys and CCD cells. FPC levels increased in r-cpk kidneys and siRNA of Cys1 in wt cells reduced FPC. However, FPC deficiency in Pkhd1 mutants did not affect cystin levels. Cystin deficiency and associated FPC loss impacted the architecture of the primary cilium, but not ciliogenesis. No reduction in Pkhd1 mRNA levels in cpk kidneys and CCD cells suggested posttranslational FPC loss. Studies of cellular protein degradation systems suggested selective autophagy as a mechanism. In support of the previously described function of FPC in E3 ubiquitin ligase complexes, we demonstrated reduced polyubiquitination and elevated levels of functional epithelial sodium channel in cpk cells. Therefore, our studies expand the function of cystin in mice to include inhibition of Myc expression via interaction with necdin and maintenance of FPC as functional component of the NEDD4 E3 ligase complexes. Loss of FPC from E3 ligases may alter the cellular proteome, contributing to cystogenesis through multiple, yet to be defined, mechanisms.

Lipopolysaccharide (LPS) serves as the interface between gram-negative bacteria (GNB) and the innate immune response in respiratory epithelial cells (REC). Herein, we describe a novel biological role of LPS that permits GNB to persist in the respiratory tract through inducing CFTR and mucociliary dysfunction. LPS reduced cystic fibrosis transmembrane conductance regulater (CFTR)-mediated short-circuit current in mammalian REC in Ussing chambers and nearly abrogated CFTR single channel activity (defined as forskolin-activated Cl- currents) in patch clamp studies, effects of which were blocked with toll-like receptor (TLR)-4 inhibitor. Unitary conductance and single-channel amplitude of CFTR were unaffected, but open probability and number of active channels were markedly decreased. LPS increased cytoplasmic and mitochondrial reactive oxygen species resulting in CFTR carbonylation. All effects of exposure were eliminated when reduced glutathione was added in the medium along with LPS. Functional microanatomy parameters, including mucociliary transport, in human sinonasal epithelial cells in vitro were also decreased, but restored with co-incubation with glutathione or TLR-4 inhibitor. In vivo measurements, following application of LPS in the nasal cavities showed significant decreases in transepithelial Cl- secretion as measured by nasal potential difference (NPD) – an effect that was nullified with glutathione and TLR-4 inhibitor. These data provide definitive evidence that LPS-generated reactive intermediates downregulate CFTR function in vitro and in vivo which results in cystic fibrosis-type disease. Findings have implications for therapeutic approaches intent on stimulating Cl- secretion and/or reducing oxidative stress to decrease the sequelae of GNB airway colonization and infection.

Cystic fibrosis (CF) is a life-shortening, genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The primary cause of CF is reduced CFTR-mediated chloride and bicarbonate transport, due to mutations in CFTR. However, inflammation and persistent infections influence clinical outcome. Cellular stress response pathways, such as the unfolded protein response (UPR) and the integrated stress response (ISR), referred to here as cellular stress response pathways (SRPs), contribute to the pathology of human disorders. Multiple studies have indicated activation of SRPs in CF tissues. We review our present understanding of how SRPs are activated in CF and their contribution to pathology. We conclude that reduced CFTR function in CF organs establishes a tissue environment in which internal or external insults activate SRPs. SRPs contribute to CF pathogenesis by reducing CFTR expression, enhancing inflammation with consequent tissue remodeling. Understanding the contribution of SRPs to CF pathogenesis is crucial even in the era of CFTR "modulators" that are designed to potentiate, correct or amplify CFTR function, since there is an urgent need for supportive treatments. Importantly, CF patients with established pathology could benefit from the targeted use of drugs that modulate SRPs to reduce the symptoms.

BACKGROUND:Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further. METHODS:In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule. RESULTS:Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions. CONCLUSION:The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo.

The cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial sodium channels (ENaC) are located in the apical membranes of airway and alveolar epithelial cells. These transporters play an important role in the regulation of lung fluid balance across airway and alveolar epithelia by being the conduits for chloride (Cl-) and bicarbonate ([Formula: see text]) secretion and sodium (Na+) ion absorption, respectively. The functional role of these channels in the respiratory tract is to maintain the optimum volume and ionic composition of the bronchial periciliary fluid (PCL) and alveolar lining fluid (ALF) layers. The PCL is required for proper mucociliary clearance of pathogens and debris, and the ALF is necessary for surfactant homeostasis and optimum gas exchange. Dysregulation of ion transport may lead to mucus accumulation, bacterial infections, inflammation, pulmonary edema, and compromised respiratory function. Influenza (or flu) in mammals is caused by influenza A and B viruses. Symptoms include dry cough, sore throat, and is often followed by secondary bacterial infections, accumulation of fluid in the alveolar spaces and acute lung injury. The underlying mechanisms of flu symptoms are not fully understood. This review summarizes our present knowledge of how influenza virus infections alter airway and alveolar epithelial cell CFTR and ENaC function in vivo and in vitro and the role of these changes in influenza pathogenesis.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to deletion of the phenylalanine at position 508 (ΔF508) in the CFTR protein and causes multiple folding and functional defects. Contrary to large-scale efforts by industry and academia, no significant therapeutic benefit has been achieved with a single “corrector”. Therefore, investigations concentrate on drug combinations. Orkambi (Vertex Pharmaceuticals), the first FDA-approved drug for treatment of cystic fibrosis (CF) caused by this mutation, is a combination of a corrector (VX-809) that facilitates ΔF508 CFTR biogenesis and a potentiator (VX-770), which improves its function. Yet, clinical trials utilizing this combination showed only modest therapeutic benefit. The low efficacy Orkambi has been attributed to VX-770-mediated destabilization of VX-809-rescued ΔF508 CFTR. Here we report that the negative effects of VX-770 can be reversed by increasing the half-life of the endoplasmic reticulum (ER) form (band B) of ΔF508 CFTR with another corrector (Corr-4a.) Although Corr-4a alone has only minimal effects on ΔF508 CFTR rescue, it increases the half-life of ΔF508 CFTR band B when it is present during half-life measurements. Our data shows that stabilization of band B ΔF508 CFTR with Corr-4a and simultaneous rescue with VX-809, leads to a >2-fold increase in cAMP-activated, CFTRinh-172-inhibited currents compared to VX-809 alone, or VX-809+VX-770. The negative effects of VX-770 and the Corr-4a protection are specific to the native I507-ATT ΔF508 CFTR without affecting the inherently more stable, synonymous variant I507-ATC ΔF508 CFTR. Our studies emphasize that stabilization of ΔF508 CFTR band B in the ER might improve its functional rescue by Orkambi.

T17M rhodopsin expression in rod photoreceptors leads to severe retinal degeneration and is associated with the activation of ER stress related Unfolded Protein Response (UPR) signaling. Here, we show a novel role of a UPR transcription factor, ATF4, in photoreceptor cellular pathology. We demonstrated a pro-death role for ATF4 overexpression during autosomal dominant retinitis pigmentosa (ADRP). Based on our results in ATF4 knockout mice and adeno-associated viral (AAV) delivery of ATF4 to the retina, we validated a novel therapeutic approach targeting ATF4 over the course of retinal degeneration. In T17M rhodopsin retinas, we observed ATF4 overexpression concomitantly with reduction of p62 and elevation of p53 levels. These molecular alterations, together with increased CHOP and caspase-3/7 activity, possibly contributed to the mechanism of photoreceptor cell loss. Conversely, ATF4 knockdown retarded retinal degeneration in 1-month-old T17M Rhodopsin mice and promoted photoreceptor survival, as measured by scotopic and photopic ERGs and photoreceptor nuclei row counts. Similarly, ATF4 knockdown also markedly delayed retinal degeneration in 3-month-old ADRP animals. This delay was accompanied by a dramatic decrease in UPR signaling, the launching of anti-oxidant defense, initiation of autophagy, and improvement of rhodopsin biosynthesis which together perhaps combat the cellular stress associated with T17M rhodopsin. Our data indicate that augmented ATF4 signals during retinal degeneration plays a cytotoxic role by triggering photoreceptor cell death. Future ADRP therapy regulating ATF4 expression can be developed to treat retinal degenerative disorders associated with activated UPR.

Synonymous mutations, such as I507-ATC -> ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct Delta F508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC Delta F508 CFTR biogenesis and function. C4 stabilized I507-ATT Delta F508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected similar to 10% of both of the variants, leading to stable, forskolin+3isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC Delta F508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC Delta F508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC Delta F508 CFTR. Our results suggest that binding of C4 and VX-809 to Delta F508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.-Bali, V., Lazrak, A., Guroji, P., Fu, L., Matalon, S., Bebok, Z. A synonymous codon change alters the drug sensitivity of Delta F508 cystic fibrosis transmembrane conductance regulator. FASEB J. 30, 201-213 (2016). www.fasebj.org