Feige Kaplan

A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Haplotypes in the Dystrophin DNA Segment Point to a Mosaic Origin of Modern Human Diversity

Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one T(n) microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the T(n) microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000-56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions.

Identification of Candidate Active Site Residues in Lysosomal β-Hexosaminidase A

The β-hexosaminidases (Hex) catalyze the cleavage of terminal amino sugars on a broad spectrum of glycoconjugates. The major Hex isozymes in humans, Hex A, a heterodimer of α and β subunits (αβ), and Hex B, a homodimer of β subunits (ββ), have different substrate specificities. The β subunit (HEXB gene product), hydrolyzes neutral substrates. The α subunit (HEXA gene product), hydrolyzes both neutral and charged substrates. Only Hex A is able to hydrolyze the most important natural substrate, the acidic glycolipid GM2 ganglioside. Mutations in the HEXA gene cause Tay-Sachs disease (TSD), a GM2 ganglioside storage disorder. We investigated the role of putative active site residues Asp-α258, Glu-α307, Glu-α323, and Glu-α462 in the α subunit of Hex A. A mutation at codon 258 which we described was associated with the TSD B1 phenotype, characterized by the presence of normal amounts of mature but catalytically inactive enzyme. TSD-B1 mutations are believed to involve substitutions of residues at the enzyme active site. Glu-α307, Glu-α323, and Glu-α462 were predicted to be active site residues by homology studies and hydrophobic cluster analysis. We used site-directed mutagenesis and expression in a novel transformed human fetal TSD neuroglial (TSD-NG) cell line (with very low levels of endogenous Hex A activity), to study the effects of mutation at candidate active site residues. Mutant HEXA cDNAs carrying conservative or isofunctional substitutions at these positions were expressed in TSD-NG cells. αE323D, αE462D, and αD258N cDNAs produced normally processed peptide chains with drastically reduced activity toward the α subunit-specific substrate 4MUGS. The αE307D cDNA produced a precursor peptide with significant catalytic activity. Kinetic analysis of enzymes carrying mutations at Glu-α323 and Asp-α258 (reported earlier by Bayleran, J., Hechtman, P., Kolodny, E., and Kaback, M. (1987) Am. J. Hum. Genet. 41, 532-548) indicated no significant change in substrate binding properties. Our data, viewed in the context of homology studies and modeling, and studies with suicide substrates, suggest that Glu-α323 and Asp-α258 are active site residues and that Glu-α323 is involved in catalysis.

Reduced Viability of Mice with Lung Epithelial-Specific Knockout of Glucocorticoid Receptor

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi⁻) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepi⁻ mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi⁻ mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; β- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.

The French Canadian Tay-Sachs disease deletion mutation: identification of probable founders

SummaryTay-Sachs disease (TSD) is an inherited neurodegenerative ganglioside storage disorder caused by deficiency of the hexosaminidase A enzyme. A deletion allele (FCD) at the HEXA locus has attained high frequency in the French Canadian population. The distribution of affected probands shows a likely center of diffusion for this mutation located in the Bas-St.-Laurent and Gaspésie regions of the province of Quebec. We have reconstructed the genealogies of 15 obligate carriers of the FCD allele to an average depth of 12 generations identifying 60 ancestors and 80 European founders common to all of them. The ancestral origins of the European founders show a significantly greater number of individuals born in the French provinces of Normandy and Perche than expected based on information regarding the origins of the 8,500 immigrants who settled the colony of New France during the French regime. We have identified common ancestors among the 10 who were born in Quebec who appear to be likely candidates for the origin of the FCD mutation. One such couple had 11 children, 5 of whom settled in regions of Quebec or New Brunswick that today have elevated heterozygote frequencies for the FCD. The five offspring are ancestors of all known carriers. By contrast, the absence of FCD alleles among TSD probands in France suggests that the mutation did not occur in a European founder.

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a G→A transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada.

Lgl1, a mesenchymal modulator of early lung branching morphogenesis, is a secreted glycoprotein imported by late gestation lung epithelial cells

Secreted glycoproteins serve a variety of functions related to cell-cell communication in developmental systems. We cloned LGL1, a novel glucocorticoid-inducible gene in foetal lung, and described its temporal and spatial localization in the rat. Disruption of foetal mesenchyme-specific LGL1 expression using antisense oligodeoxynucleotides, which was associated with a 50% decrease in lgl1 protein levels, inhibited airway epithelial branching in foetal rat gestational day 13 lung buds in explant culture. These findings suggested that lgl1 functions as a secreted signalling molecule. We now provide evidence supporting a role for lgl1 in mesenchymal-epithelial interactions that govern lung organogenesis. Lgl1 is a secreted glycoprotein with a conserved N-terminal secretory signal peptide. Using dual immunofluorescence, intracellular lgl1 was found to co-localize with markers of the Golgi apparatus and endoplasmic reticulum, consistent with its association with secretory vesicles. Using pulse-chase studies, we show that lgl1 is a stable protein with a half-life of 11.5 h. Furthermore, at gestational days 20 and 21 (term=22), foetal distal lung epithelial cells import lgl1 protein. Taken together, our findings support distinct roles for lgl1 as a mediator of glucocorticoid-induced mesenchymal-epithelial interactions in early and late foetal lung organogenesis.

Importin-13 genetic variation is associated with improved airway responsiveness in childhood asthma

BackgroundGlucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20).Methods10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association.ResultsAmong participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51–2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01–0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids.ConclusionIPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids.

Interaction between importin 13 and myopodin suggests a nuclear import pathway for myopodin

Importin 13 is a member of the importin β superfamily of nuclear transport proteins and is expressed in multiple tissues at high levels both in humans and rodents, including fetal lung, brain, and heart. In order to elucidate potential functions of imp13 in the heart, we have used rat imp13 as bait to screen a human heart cDNA library and identified an interaction with the C-terminal peptide of myopodin (a.a. 360–698), an actin-bundling protein, associated with tumor–suppressor activity that localizes to both the cytoplasm and the nucleus. We have used GST-pull down assays and co-immunoprecipitation experiments to demonstrate an interaction between imp13 and full-length myopodin and observed that RanGTP dissociates the myopodin–imp13 complex. In studies of cultured cells, we show that both imp13 siRNA and a C-terminal fragment of imp13 protein prevent nuclear localization of myopodin. We, therefore, conclude that imp13 functions in myopodin import and we suggest that the regulation of these events is critical for normal and abnormal cellular differentiation.

Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice

BackgroundNeonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.MethodsAn Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.ResultsAbsence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.ConclusionOur findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.