Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.     

Mesenchymal control of branching pattern in the fetal mouse lung

The effect of mesenchyme on specialization of respiratory epithelium in the fetal mouse was tested in organ cultures. Heterologous combinations were made between respiratory and non-respiratory lung epithelia and the corresponding mesenchymes. Isolated terminal respiratory buds of fetal mouse lungs were recombined with mesenchyme from chick lung parabronchi, mouse trachea or from the avascular, non-respiratory air sacs of chick lungs. Isolated non-branching chick air sacs were combined with mouse terminal bud mesenchyme or mesenchyme from the respiratory branches of chick lungs. Air sac epithelia branched in a pattern characteristic of the chick lung when combined with chick respiratory mesenchyme and in a pattern characteristic of mouse lung when combined with mouse terminal bud mesenchyme. Mouse terminal bud epithelia did not branch with either mouse tracheal mesenchyme or chick air sac mesenchyme but branched in a chick pattern with chick parabronchial mesenchyme. Electron microscopic examination of the cultures showed that all chick air sac epithelial cultures failed to produce surfactant (lamellar bodies) even when they branched. Control cultures of mouse terminal buds contained large numbers of lamellar bodies; mesenchyme which suppressed branching reduced the number of lamellar bodies to only a few in a small proportion of the cells. Culture medium supplemented with growth factors and hormones increased the number of lamellar bodies in heterologous mouse combinations but did not bring the number to control levels. Supplemented medium had no effect on lamellar body production by chick air sac epithelium. The results indicate that branching pattern is determined by the mesenchyme surrounding the epithelial primordium. However, the capacity to synthesize surfactant is determined by the source of the epithelium; mesenchyme may control the degree of expression but not the absolute presence or absence of the differentiated condition.     

Use of lipoplex-induced nuclear factor-kappaB activation to enhance transgene expression by lipoplex in mouse lung

BACKGROUND: Although lipofection-induced TNF-alpha can activate nuclear factor kappaB (NF-kappaB), which, in turn, increases the transgene expression from plasmid DNA in which any NF-kappaB responsive element is incorporated, no attempts have been made to use such biological responses as NF-kappaB activation against a vector to enhance vector-mediated gene transfer. METHODS: A lipoplex composed of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium and cholesterol liposome and plasmid DNA encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pCMV-Luc) was intravenously injected into mice. Luciferase activity as well as NF-kappaB activation in the lung were evaluated. Then, a novel plasmid DNA, pCMV-kappaB-Luc, was constructed by inserting 5 repeats of NF-kappaB-binding sequences into the pCMV-Luc. RESULTS: NF-kappaB in the lung was activated by injection of the lipoplex and its nuclear localization was observed. An injection of lipopolysaccharide 30 min prior to the lipofection further activated NF-kappaB. At the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and NF-kappaB activity. Based on these findings, we tried to enhance the lipoplex-based transgene expression by using NF-kappaB activation. The lipoplex consisting of pCMV-kappaB-Luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pCMV-Luc. CONCLUSIONS: We demonstrated that NF-kappaB activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting NF-kappaB-binding sequences into plasmid DNA. These findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.     

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells

BackgroundPulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells.MethodsWe examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated.ResultsVEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-α antibody to the medium.ConclusionsThe protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-α released from papain-treated lung cells induces MSC to secret VEGF-A.     

Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei

The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complex-lamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. 5-day-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear 'activity'.     

Collagen involvement in branching morphogenesis of embryonic lung and salivary gland

The possibility that extracellular collagen is involved in branching morphogenesis of mouse embryo lung and salivary glands has been explored duringin vitro organ culture. Control cultures of both rudiment types contain abundant collagen in extracellular spaces between mesenchymal cells and in the epithelial-mesenchymal interface. Branching morphogenesis of lungs and salivary glands is not perturbed by the presence of β-aminopropionitrile, implying that extracellular collagen cross-linking is not required, but is perturbed by α,α′-dipyridyl orl-azetidine-2-car?ylic acid (LACA), agents reported to interfere with collagen synthesis and secretion. Analysis of the structural and biosynthetic effects of LACA revealed a severe inhibition of collagen synthesis, as monitored by hydroxyproline synthesis, and extracellular collagen accumulation. Cell and tissue integrity was not affected, but a slight inhibition of general protein synthesis, protein accumulation, and epithelial expansion was observed. The strong correlations between collagen biosynthesis, extracellular collagen presence, and branching morphogenesis are consistent with an integral role for collagen in embryonic lung and salivary gland morphogenesis.     

Ailanthus altissima swingle has anti-anaphylactic effect and inhibits inflammatory cytokine expression via suppression of nuclear factor-kappaB activation

Ailanthus altissima swingle (ailanthic cortex, AAS) has been used as a traditional medicine for fever, bleeding, infection, and inflammation for many years in Korea. However, its mechanisms have not been examined. In the present study, we investigate the effect of AAS on the mast-cell-mediated allergic and inflammatory reaction using in vivo and in vitro models and elucidate its molecular mechanisms. AAS significantly inhibited compound 48/48-induced edema and systemic anaphylaxis. AAS significantly inhibited passive cutaneous anaphylaxis. AAS inhibited histamine release from rat peritoneal mast cells (RPMCs) in a dose-dependent manner. Moreover, AAS significantly inhibited production of inflammatory cytokines, tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 on the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated human mast cell line, HMC-1 cells. AAS inhibits the IgE or stem cell factor-induced TNF production on RPMCs. In activated HMC-1 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus, whereas the level of NF-kappaB/Rel A in the nucleus was decreased by AAS treatment. In addition, AAS inhibited the PMACI-induced IkappaBalpha degradation. In conclusion, the present results indicate that AAS has potent anti-anaphylactic and anti-inflammatory properties.     

Fhit protein inhibits cell growth by attenuating the signaling mediated by nuclear factor-kappaB in colon cancer cell lines

Fragile histidine triad (FHIT) gene is involved in the deletions at the 3p14.2 region in various cancers. We investigated the role of Fhit protein in cell growth by examining the signaling pathway affected by Fhit. We used 3 human colon cancer cell lines, SW480, DLD-1 and COLO201, in the study. SW480 cells, in which the expression of Fhit is completely absent, were transfected with pIRES1neo vector (SW/IRES cells), wild-type FHIT vector (SW/FHIT cells) or mt-FHIT (codon 96, His changed to Asn) vector (SW/mt-FHIT cells). The growth of SW/FHIT or SW/mt-FHIT cells was suppressed in comparison with that of parent or SW/IRES cells. Especially, the growth of SW/FHIT cells was considerably suppressed. On the other hand, the silencing of FHIT by an siRNA for it in SW/FHIT or DLD-1 cells harboring Fhit demonstrated that the growth of FHIT siRNA-treated cells was significantly enhanced in comparison with that of the vector control or nonspecific siRNA control. Thus, we found that Fhit negatively contributed to cell growth in the colon cancer cell lines. Moreover, SW/FHIT cells exhibited a higher sensitivity to oxidative stress evoked by inhibitors of mitochondrial electron transport or proteasomes compared with any of the control transfectants. The base line amount of phospho-IkappaB-alpha (p-IkappaB-alpha) was reduced in SW/FHIT cells compared with that in the other transfectants. On the contrary, the FHIT siRNA-treated SW/FHIT and DLD-1 cells exhibited an elevated p-IkappaB-alpha level in an RNAi experiment on FHIT. Perturbation of nuclear factor (NF)-kappaB signaling was strongly suggested by the fact that the wild-type Fhit expressants of SW480 cells tended to be sensitive to sulfasarazine or parthenolide, which are inhibitors of NF-kappaB. The time course of the level of IkappaB kinase (IKK) complex (IKKalpha/beta, phospho-IKKalpha/beta and IKKgamma) after the treatment with TNF-alpha was similar between the transfectants. Although p-IkappaB-alpha and phospho-NF-kappaB p65 (p-NF-kappaB) in SW/FHIT cells responded to TNF-alpha as those in other transfectants, the increase in the levels of p-IkappaB-alpha and p-NF-kappaB after a 5-min treatment was less in SW/FHIT cells than in the other transfectants. These results altogether suggest that Fhit functions as an anti-oncoprotein by inhibiting the phosphorylation of IkappaB-alpha and thereby blocking NF-kappaB signaling.     

Paralysis and growth of the musculoskeletal system in the embryonic chick

Avian embryos can be completely paralyzed by injection of neuromuscular-blocking agents. We used a single injection of decamethonium iodide to paralyze embryos at 7, 8, or 10 days of incubation and analyzed the growth of individual bones (clavicle, mandible, ulna, femur, tibia, humerus) and of individual muscles that act upon some of those bones (clavicular and sternal heads of m. pectoralis, and mm. biceps brachii, depressor mandibulae, pseudotemporalis, and adductor externus). Growth of the bones is not equally affected by paralysis. Only 27% of clavicular growth (by mass) but 77% of mandibular growth occurred in paralyzed embryos, whereas the four long bones exhibited 52-63% of their normal growth. Analysis of muscle weight, fiber length and physiological cross-sectional area (weight/fiber length) indicate that there was greater reduction of the muscles acting on the limbs than of those acting on the mandible, i.e., diminished growth of the skeleton is correlated with reduced muscular activity. Specific retardation of clavicular growth is due to fusion of sternal rudiments and collapse of the thorax, as well as virtual absence of the musculature that normally attaches to the clavicle. We discuss these results in the light of intrinsic and extrinsic factors governing growth of the embryonic skeleton. Paralysis reduces skeletal growth by reducing both the movements taking place in ovo, and the loads imposed on the bones by muscle contraction, changes that represent alterations in the mechanical environment of the skeleton.     

Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate

 Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5–15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell–cell communication, cell fate, and differentiation of conducting airway epithelia. Accepted: 5 May 1997     

Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.     

CEPU-1 expression in the early embryonic chick brain

We describe the expression pattern of CEPU-1, a cell adhesion molecule of the immunoglobulin superfamily, in the early chick embryo brain. An initially broad domain of expression, encompassing forebrain, midbrain and anterior hindbrain, is subsequently narrowed down to a ring-shaped domain at the midbrain-hindbrain boundary, co-localizing precisely with the expression of Wnt1 at the isthmus. In addition, CEPU-1 is expressed in the dorsal aspect of rhombomere 4 and its emigrating neural crest cells. Later in development, we also find CEPU-1 expression in other parts of the developing nervous system such as sensory ganglia and in the ventral aspect of forebrain, midbrain and hindbrain.