Expression analysis of the histamine H(3) receptor in developing rat tissues.

Endogenous histamine is involved in tissue growth and cell proliferation. In accordance with a putative function of the H(3) receptor in this mitogenic effect, we show that H(3)-receptor mRNAs are expressed together with those of the histamine-synthesizing enzyme in the embryonic liver and adipose tissue, and in various epithelia. Finally, we show that activation of recombinant H(3) receptors enhances MAP kinase activity.     

Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells.

The ABCA3 gene, of the ABCA subclass of ATP-binding cassette (ABC) transporters, is expressed exclusively in lung. We report here the cloning, molecular characterization, and distribution of human ABCA3 in the lung. Immunoblot analysis using the specific antibody reveals a 150-kDa protein in the crude membrane fraction of human lung. Immunohistochemical analyses of alveoli show that ABCA3 is expressed only in the type II cells expressing surfactant protein A. At the ultrastructural level, ABCA3 immunoreactivity was detected mostly at the limiting membrane of the lamellar bodies. Since members of the ABCA transporter family are known to be involved in transmembrane transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant in type II cells.     

Expression of a cardiac myosin gene in non-heart tissues of developing frogs

Direct developing frogs, like Eleutherodactylus coqui, provide opportunities to investigate limb early development in anuran amphibians that are less available in species with tadpoles. We have found that myosin heavy chain 6 (myh6), a myosin gene usually considered heart-specific in Xenopus and other animals, is expressed in limbs of E. coqui embryos. The gene for microRNA(miR)-208 is contained in an intron of the E. coqui myh6 gene as in mammals, and miR -208 was detected as a microRNA, more highly expressed in a microarray of E. coqui limb buds, compared to Xenopus laevis limb buds. Myh6 is also expressed in several muscles of tadpoles and froglets of Xenopus tropicalis. These connections raise the possibility of an involvement of myh6 and miR-208 in the thyroid dependent metamorphosis of anurans.     

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound beta-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophila galectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophila galectin had a unique and specific tissue distribution. Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.     

Enhanced 3-methoxytyramine levels in crown gall tumours and other undifferentiated plant tissues.

An amine, after dansylation, has been isolated from Nicotiana tabacum crown gall tumours for the first time and characterized as 4-hydroxy-3-methoxy-beta-phenylethylamine (3-methoxytyramine). The compound cannot be detected in differentiated N. tabacum tissues but appears in the corresponding callus controls. Its concentration is further increased 5-42-fold when N. tabacum is transformed with all strains of Agrobacterium tumefaciens so far tested.     

Expression of Tcf/Lef and sFrp and localization of beta-catenin in the developing mouse lung.

Recent evidence that Wnts and other genes in the Wnt signaling pathway are expressed in embryonic and adult mouse lung suggests that this pathway is important for cell fate decisions and differentiation of lung cell types. We therefore examined the expression and protein distribution of several Wnt pathway components during prenatal mouse lung development using whole-mount in situ hybridization and immunohistochemistry. Between embryonic days 10.5 and 17.5 (E10.5-E17.5), beta-catenin was localized in the cytoplasm, and often also the nucleus, of the undifferentiated primordial epithelium (PE), differentiating alveolar epithelium (AE; present from E14.5 onward), and adjacent mesenchyme. Tcf1, Lef1, Tcf3, Tcf4, sFrp1, sFrp2 and sFrp4 were also expressed in the PE, AE, and adjacent mesenchyme in specific spatio-temporal patterns.     

Expression of intermediate filament proteins in fetal and adult human lung tissues

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.     

Expression of lipoprotein lipase associated with lung adenocarcinoma tissues

Lipoprotein lipase (LPL) plays a key role in the lipid metabolism and transporting. It can catalyze the hydrolysis of chylomicron and very low-density lipoprotein triglyceride. Moreover, the abnormality of LPL associates with many pathophysiological conditions. Herein cDNA microarray and Northern blots analysis were used to study the expression of lipoprotein lipase in lung adenocarcinoma tissues. There were 113 genes of all tested blots in cDNA microarray expressed lowly. LPL gene is expressed lowly at the average ratio 0.26 (Cy5/Cy3) in lung adenocarcinoma tissues over controls. Northern blots confirmed those changes detected from the cDNA microarray and suggested that low expression of LPL may play an important role in the lung adenocarcinoma development.     

Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man

Adrenocorticotropin-releasing hormone (CRH) is a peptide originally isolated from the hypothalamus. Immunocytochemical and RIA studies have revealed that CRH-like peptide is also localized in human nonhypothalamic tissues and some tumors. To see if CRH is synthesized in these nonhypothalamic tissues and tumors, we examined preproCRH mRNA in these tissues by Northern blot analysis using a cloned human preproCRH gene as a probe. PreproCRH mRNA was detected in human hypothalamus, cerebral cortex, adrenal gland, placenta, pheochromocytoma, and thymic carcinoid. The content of preproCRH mRNA in placenta was apparently greater than that in the whole hypothalamus.     

Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

Background

ABCA3 transporter (ATP-binding cassette transporter of the A subfamily) is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII). It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD) of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER).

Methods

Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level.

Results

We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C) or complete (L101P) retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling.

Conclusion

Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that might contribute to the pathogenesis of genetic ILD.     

Expression of autotaxin and lysophosphatidic acid receptors 1 and 3 in the developing rat lung and in response to hyperoxia

Abstract

We sought to evaluate lysophosphatidic acid (LPA) signaling improvement in lung development by assessing the expression of autotaxin and LPA receptor 1 and 3 (LPAR1 and LPAR3) in the neonatal rat lung during normal perinatal development and in response to hyperoxia. In the developmental study, rats were sacrificed on days 17, 19, and 21 of gestation; on postnatal days 1, 4, and 7; and at adulthood (postnatal 9 weeks). In the hyperoxia study, 42 postnatal 4-day-old rat pups were divided into seven groups and exposed to either 85% O₂ for 24, 72, or 120 h or room air for 0, 24, 72, or 120 h. The rats were then euthanized after 0, 24, 72, and 120 h of exposure. Immunofluorescence demonstrated that autotaxin, LPAR1, and LPAR3 proteins were broadly colocalized in airway epithelial cells, but mainly distributed in vascular endothelial and mesenchymal cells during the first postnatal week. The expression of autotaxin, LPAR1, and LPAR3 were increased during late gestation and then decreased after birth. Autotaxin expression and enzymatic activity were significantly increased at 72 and 120 h after exposure to hyperoxia. LPAR1 and LPAR3 expression was also increased after 120 h of hyperoxic exposure. These findings suggest that LPA-associated molecules were upregulated at birth and induced by hyperoxia in the developing rat lung. Therefore, the LPA pathway may be involved in normal lung development, including vascular development, as well as wound-healing processes of injured neonatal lung tissue, which is at risk of neonatal hyperoxic acute lung injury.     

Expression of SHH signaling pathway components in the developing human lung

The Sonic hedgehog (Shh) cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In the present study, the expression patterns of SHH signaling pathway components, including SHH, PTCH1, SMO, GLI1, GLI2 and GLI3 were examined by in situ hybridization and immunohistochemistry, and compared with the equivalent patterns in mice. Our results showed that, as in mice, SHH was expressed in the epithelium of the developing human lung. However, SHH receptors (PTCH1 and SMO) and SHH signaling effectors (GLI1-3) were strongly detected in the human lung epithelium, but weakly in the mesenchyme, slightly different from their expressions in mice. Furthermore, the expression levels of SHH signaling pathway genes in human lung, but not that of GLI1, were subsequently downregulated at the canalicular stage evaluated by real-time PCR, coincident with a decline in the developing murine lung. In conclusion, in spite of slight differences, the considerable similarities of gene expression in human and mice suggest that conserved molecular networks regulate mammalian lung development.