Membrane Mucin Muc4 promotes blood cell association with tumor cells and mediates efficient metastasis in a mouse model of breast cancer.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4ko/NDL female mice exhibit similar latencies and growth rates as Muc4wt/NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4ko/NDL mice. Interestingly, histological analysis of lung lesions from Muc4ko/NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4ko/NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4wt/NDL cells than Muc4ko/NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

The ER stress transducer IRE1ß is required for airway epithelial mucin production.

Inflammation of human bronchial epithelia (HBE) activates the endoplasmic reticulum (ER) stress transducer inositol-requiring enzyme 1 (IRE1)α, resulting in IRE1α-mediated cytokine production. Previous studies demonstrated ubiquitous expression of IRE1α and gut-restricted expression of IRE1ß. We found that IRE1ß is also expressed in HBE, is absent in human alveolar cells, and is upregulated in cystic fibrosis and asthmatic HBE. Studies with Ire1ß(-/-) mice and Calu-3 airway epithelia exhibiting IRE1ß knockdown or overexpression revealed that IRE1ß is expressed in airway mucous cells, is functionally required for airway mucin production, and this function is specific for IRE1ß vs. IRE1α. IRE1ß-dependent mucin production is mediated, at least in part, by activation of the transcription factor X-box binding protein-1 (XBP-1) and the resulting XBP-1-dependent transcription of anterior gradient homolog 2, a gene implicated in airway and intestinal epithelial mucin production. These novel findings suggest that IRE1ß is a potential mucous cell-specific therapeutic target for airway diseases characterized by mucin overproduction.

Endoplasmic reticulum stress in chronic obstructive lung diseases.

Chronic airway inflammation characterizes several airway diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). The altered airway milieu that results from the pathogenic processes in these disorders affects the airway epithelia, leading to an up-regulation of their innate defense. In human airway epithelia, luminal inflammatory stimuli induce an adaptation characterized by an expansion of the endoplasmic reticulum (ER) and its Ca(2+) stores. This epithelial adaption mediates Ca(2+)-dependent "hyperinflammatory" responses, and recent studies have shown that activation of the unfolded protein response (UPR) by ER stress is involved in the process. The UPR is also known to be activated by cigarette smoke, the primary trigger for development of COPD. These studies illustrate the functional role of UPR pathways during airway inflammation and suggest that targeting the UPR may be a therapeutic strategy for obstructive airway diseases. This article reviews the link between airway epithelial inflammation and activation of the UPR, and discusses how UPR activation might be relevant for CF and COPD airways disease.

Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.

It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⁺ mice, airway-targeted overexpression of the epithelial Na⁺ channel ß subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⁺ mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⁺ mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll-interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⁺ mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⁺ mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⁺ mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.

Transgenic hCFTR expression fails to correct ß-ENaC mouse lung disease.

The relationships between airway epithelial Cl(-) secretion-Na(+) absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl(-) secretion and regulated Na(+) absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (∼2.5-fold) elevated basal Cl(-) secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na(+) absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the ß-subunit of the epithelial Na(+) channel (ß-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/ß-ENaC transgenic mouse airways, which exhibited an elevated basal Cl(-) secretion and Na(+) hyperabsorption. However, the airway disease characteristic of ß-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of ∼6 µm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 µm in ß-ENaC and CCSP-hCFTR/ß-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl(-) secretion but does not regulate Na(+) transport in WT mice and 2) transgenic hCFTR produces increased Cl(-) secretion, but not regulation of Na(+) channels, in ß-ENaC mouse airways and does not ameliorate ß-ENaC mouse lung disease.

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice.

Successful gene therapy will require that the therapeutic gene be expressed at a sufficient level in the correct cell type(s). To improve the specificity of gene transfer for cystic fibrosis (CF) and other airway diseases, we have begun to develop cell-type specific promoters to target the expression of transgenes to specific airway cell types. Using a FOXJ1 promoter construct previously shown to direct transgene expression specifically to ciliated cells, we have generated transgenic mice expressing human cystic fibrosis transmembrane conductance regulator (CFTR) in the murine tracheal and nasal epithelia. RNA analysis demonstrated levels of CFTR expression is greater than or equal to the level of endogenous mouse CFTR. Immunoprecipitation and western blotting demonstrated the production of human CFTR protein, and immunochemistry confirmed that CFTR was expressed in the apical region of ciliated cells. The transgenic animals were bred to CFTR null mice (Cftr(tm1Unc)) to determine if expression of CFTR from the FOXJ1 promoter is capable of correcting the airway defects in Cl(-) secretion and Na(+) absorption that accompany CF. Isolated trachea from neonatal CF mice expressing the FOXJ1/CFTR transgene demonstrated a correction of forskolin-stimulated Cl(-) secretion. However, expression of human CFTR in ciliated cells of the nasal epithelia failed to significantly change the nasal bioelectrics of the CF mice.

Cloning and functional characterization of two murine uridine nucleotide receptors reveal a potential target for correcting ion transport deficiency in cystic fibrosis gallbladder.

Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y(2) receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y(2) receptor gene disruption. Pharmacological data suggested the involvement of P2Y(4) and P2Y(6) receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y(4) and P2Y(6) receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca(2+) mobilization. The (m)P2Y(4) receptor was potently, but not selectively, activated by UTP (UTP > or = ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y(6) receptor was highly selective for UDP (UDP >> ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y(4) and (m)P2Y(6) receptors resemble those for nucleotide-promoted chloride transport in murine P2Y(2)(-/-) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (I(sc)) was impaired in the CF mouse, UDP-promoted I(sc) changes were not affected in CF gallbladder epithelium, suggesting that the P2Y(6) receptor is a target for treatment of CF gallbladder disease.

Multiple advantages of alpha-fetoprotein as a marker for in vivo gene transfer.

The development of improved gene transfer vectors has been hampered by the lack of a nonimmunogenic reporter gene that can be serially quantified in the serum or from other sites. In response to the need to develop a new reporter protein, we have evaluated alpha-fetoprotein (AFP) as a potential candidate. A first-generation E1/E3-deleted adenoviral vector expressing human AFP (hAFP) was generated as a preliminary tool to evaluate AFP as a reporter. Using both mouse and baboon models, hAFP expression was evaluated in serum after intravenous delivery and in serum and bronchioalveolar lavage (BAL) fluid after delivery to the lung. In immunocompetent animals, intravenous delivery of the hAFP adenoviral vector resulted in hAFP expression in the serum early after injection, which declined rapidly over time. Disappearance of hAFP from the serum was complete by 3-4 weeks after administration and was accompanied by robust antibody responses to hAFP and loss of infected cells. After lung delivery, hAFP could be detected in both serum and BAL. This allowed the analysis of the kinetics of gene expression in the lung without sacrificing the animals. In both liver and lung, immunohistochemical analysis correlated well with hAFP levels as detected in serum or BAL, indicating that serum levels were a reliable marker of tissue expression. Preliminary results with a mouse AFP expressed in a helper-dependent adenoviral vector indicate that use of a species-specific version of AFP will eliminate the complication of antibody development. These initial evaluations suggest that AFP is useful as a reporter gene to evaluate gene expression of therapeutic cassettes in multiple tissues, and it should be considered for use in human subjects.

Toxicity associated with repeated administration of first-generation adenovirus vectors does not occur with a helper-dependent vector.

BACKGROUND: Certain gene therapy protocols may require multiple administrations of vectors to achieve therapeutic benefit to the patient. This may be especially relevant for vectors such as adenoviral vectors that do not integrate into the host chromosome. Because immunocompetent animal models used for gene transfer studies develop neutralizing antibodies to adenoviral vectors after a single administration, little is known about how repeat administrations of vectors might affect transgene expression and vector toxicity. MATERIALS AND METHODS: We used mice deficient in the membrane spanning region of immunoglobulin (IgM), which do not develop antibodies, to evaluate the effect of repeated intravenous administration of first-generation and helper-dependent adenoviral vectors expressing human alpha 1-antitrypsin (hAAT). The duration and levels of transgene expression were evaluated after repeated administration of vectors. Toxicity was assessed by measuring the level of liver enzymes in the serum and the degrees of hepatocyte hypertrophy and proliferation. RESULTS: We found that previous administration of first-generation adenoviral vectors can alter the response to subsequent doses. These alterations included an increase in transgene expression early (within 1 and 3 days), followed by a rapid drop in expression by day 7. In addition, previous administrations of first-generation vectors led to an increase in toxicity of subsequent doses, as indicated by a rise in liver enzymes and an increase in hepatocyte proliferation. In contrast to first-generation vectors, use of the helper-dependent adenovirus vector, Ad-STK109, which contained no viral coding regions, did not lead to increased toxicity after multiple administrations. CONCLUSIONS: We conclude that the response of the host to adenoviral vectors can be altered after repeated administration, compared with the response after the initial vector dose. In addition, these experiments provide further evidence for the relative safety of helper-dependent adenoviral vectors for gene therapy, compared with first-generation vectors.

NSP4 elicits age-dependent diarrhea and Ca(2+)mediated I(-) influx into intestinal crypts of CF mice.

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediated ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contributing to high rates of mortality. We investigated whether infectious mammalian rotavirus, the recently characterized rotaviral enterotoxin protein NSP4, or its active NSP4(114-135) peptide, can overcome these gastrointestinal complications in CF (CFTR(m3Bay) null mutation) mice. All three agents elicited diarrhea when administered to wild-type (CFTR(+/+)), heterozygous (CFTR(+/-)), or homozygous (CFTR(-/-)) 7- to 14-day-old mouse pups but were ineffective when given to older mice. The diarrheal response was accompanied by non-age-dependent intracellular Ca(2+) mobilization within both small and large intestinal crypt epithelia. Significantly, NSP4 elicited cellular I(-) influx into intestinal epithelial cells from all three genotypes, whereas both carbachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR(-/-) background. This unique plasma membrane halide permeability pathway was age dependent, being observed only in mouse pup crypts, and was abolished by either the removal of bath Ca(2+) or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age- and Ca(2+)-dependent plasma membrane anion permeability distinct from CFTR. Furthermore, these results highlight the potential for developing synthetic analogs of NSP4(114-135) to counteract chronic constipation/obstructive bowel syndrome in CF patients.

Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery.

Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1. To test these hypotheses further, we have developed an E2a-deleted adenoviral vector expressing human alpha1-antitrypsin (hAAT). Toxicity of first-generation and E2a-deleted vectors, as determined by hematological indices, liver function tests, and histological analyses, was evaluated in C3H mice for 21 days after vector administration at increasing doses starting at 1 x 10(12) particles/kg. Both vectors induced dose-dependent abnormalities including transient thrombocytopenia, elevated ALT levels in serum, and increased hepatocyte proliferation followed by inflammation and then hypertrophy. Differences in the ratio of particles to plaque-forming units among vector preparations led to differences in hAAT expression at similar particle doses. There were no differences in toxicity between the two vectors when measured at matching levels of hAAT expression. However, the E2a-deleted vector was demonstrated to have slightly reduced hepatocyte toxicity at an intermediate particle dose. This suggests that hepatocyte toxicity is related primarily to viral entry and expression, rather than to the presence of noninfectious particles, and implies that vectors with complete elimination of viral gene expression, such as vectors with all viral coding sequences deleted, are likely to have substantial advantages in terms of safety and toxicity.

Severe phenotype in mice with termination mutation in exon 2 of cystic fibrosis gene.

Mice with a termination codon mutation in exon 2 of the cystic fibrosis (CF) gene were generated using homologous recombination in embryonic stem cells. Animals homozygous for the mutant allele display a severe intestinal phenotype similar to that previously reported for CF mutant mice. The null nature of this allele was demonstrated by the absence of detectable wild-type mRNA, by the absence of detectable CFTR in the serous gland collecting ducts of salivary tissues, and by the lack of cAMP-mediated short-circuit current responses in colonic epithelium of mutant animals.

Somatic gene therapy for cystic fibrosis.

There is considerable potential to ameliorate the pulmonary disease in cystic fibrosis (CF) using somatic gene therapy. Even low levels of expression of the gene in airways epithelium may be beneficial. Adenoviral vectors, DNA-liposome complexes, adeno-associated viral vectors and DNA-ligand complexes have been used effectively in vitro and have been tested in animals to varying extent. Adenoviral vectors and DNA-liposome complexes are being used to deliver the CF gene to patient airways in phase I clinical trials. Transient correction of the electrophysiological defect in human CF nasal epithelium has been achieved. Major goals are (i) to demonstrate that expression of the CF gene in airways epithelium will ameliorate lung disease in CF patients, and (ii) to achieve long-term expression of the introduced gene either through a single delivery with persistent expression or through the ability to use a delivery system repetitively with safety and efficacy.

A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus.

To develop an animal model for cystic fibrosis (CF), targeted gene disruption in embryonic stem (ES) cells was used to generate a duplication of exon 3 (cftrm1Bay allele) of the mouse CF gene. ES cells containing this mutation were used to generate chimeric animals that transmitted the mutant allele through the germline. Homozygous mutant animals display a severe phenotype, with approximately 40% dying within 1 week from intestinal obstruction. RNAase protection analysis of the cftrm1Bay allele did not detect any normal mRNA (< 1-2% of wild-type) in mutant animals. Pathologic changes in the intestines from mutant mice included mucus accumulation in the crypts and intestinal lumen, dilatation of the bases of the crypts, enlargement of goblet cells, and the presence of concretions in the crypts or between the villi. Changes were also present in the mucosal glands of the pharynx and the minor sublingual glands, where dilatation of acini and accumulation of eosinophilic material were evident. Atrophy of acinar cells that may be secondary to nutritional deficiency and mild inflammation in the main pancreatic duct were present in the pancreas of mutant animals. No changes were noted in the lung, trachea, liver, or male reproductive tract of mutant animals, and mutant males were fertile. Homozygous mutant mice showed defects in cAMP-mediated ion transport both in ileum and in cultured fetal tracheal explants. Thus, an additional mouse model for CF has been generated that should prove useful for the understanding of the pathogenesis and the development of treatments for CF.