Matrix GLA protein modulates branching morphogenesis in fetal rat lung

The regulation of matrix gamma-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Protein phosphatase inhibitors activate anti-fungal defence responses of soybean cotyledons and cell cultures

The application of a variety of structurally different protein phosphatase inhibitors (okadaic acid, acanthifolicin, microcystins, nodularin, tautomycin, calyculin A, cantharidin and endothall) to cut surfaces of soybean cotyledons (Glycine max L.) resulted in the production of isoflavonoid phytoalexins (plant defence compounds). Daidzein was the predominant isoflavonoid produced by soybean cotyledons in response to protein phosphatase inhibitors. In contrast, several isoflavonoid phytoalexins were seen after application of either an elicitor β-glucan fraction isolated from yeast extract or hepta-(1→3, 1→6)-β-glucoside which is the most potent elicitor-active component isolated from the soybean pathogen Phytophthora megasperma f. sp. glycinea. Isoflavonoid production in response to either protein phosphatase inhibitors or elicitors reached a maximum after 20–24 h. The addition of protein phosphatase inhibitors to a soybean cell suspension culture induced the expression of phenylalanine ammonia-lyase (PAL), the first enzyme in the isoflavonoid biosynthetic pathway. Induction of PAL activity was blocked by protein synthesis inhibitors, cycloheximide or anisomysin, and largely prevented by a protein kinase inhibitor, K252a. Another common response of plant cells to fungal elicitation, alkalinization of the soybean cell culture media, was induced within minutes in response to protein phosphatase inhibitors and was largely prevented by K252a. These studies suggest a direct role for phosphorylation in activation of plasma membrane ion flux(es), whereas the longer-term effects of protein phosphatase inhibitors on isoflavonoid production and PAL expression could be due to either direct effects of increased protein phosphorylation, or the secondary consequences of other phosphorylation-induced cellular changes. They also indicate that protein phosphatase inhibitors are likely to be of general use in investigating mechanisms of plant responses to environmental stimuli.     

Prolonged mechanical ventilation induces cell cycle arrest in newborn rat lung

Rationale

The molecular mechanism(s) by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown.

Objective

To determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats.

Methods

Seven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg⁻¹).

Measurement and Main Results

Ventilation for 24 h (h) decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation) was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8–24 h of ventilation, while that of p27^Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57^Kip2, decreased that of p16^INK4a, while the levels of p21^Waf/Cip1 and p15^INK4b were unchanged. Increased p27^Kip1 expression coincided with reduced phosphorylation of p27^Kip1 at Thr¹⁵⁷, Thr¹⁸⁷ and Thr¹⁹⁸ (p<0.05), thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg⁻¹) and when fetal lung epithelial cells were subjected to a continuous (17% elongation) cyclic stretch.

Conclusion

This is the first demonstration that prolonged (24 h) of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G₁ and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27^Kip1, p57^Kip2) from the Kip family.     

MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs

The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.     

A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.     

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.     

Modelling in vitro lung branching morphogenesis during development

It has been shown experimentally that lung epithelial explants have an ability to undergo branching morphogenesis without mesenchyme. However, the mechanisms of this phenomenon remain to be elucidated. In the present study, we construct a mathematical model that can reproduce the dynamics of in vitro branching morphogenesis. We show that the system is essentially governed by three variables--c(0) which is the initial fibroblast growth factor (FGF) concentration, D which is the diffusion coefficient of FGF, and beta which describes the mechanical strength of the cytoskeleton. It is confirmed by numerical simulations that this model can reproduce the experimentally obtained patterns qualitatively. Finally, we experimentally verify two predictions from the model: effects of very high FGF concentration and effects of small mechanical contributions of the cytoskeleton. The theoretical predictions match well with the experimental results.     

A mechanism for early branching in lung morphogenesis

The lung is a highly branched fluid-filled structure, that develops by repeated dichotomous branching of a single bud off the foregut, of epithelium invaginating into mesenchyme. Incorporating the known stress response of developing lung tissues, we model the developing embryonic lung in fluid mechanical terms. We suggest that the repeated branching of the early embryonic lung can be understood as the natural physical consequence of the interactions of two or more plastic substances with surface tension between them. The model makes qualitative and quantitative predictions, as well as suggesting an explanation for such observed phenomena as the asymmetric second branching of the embryonic bronchi.     

aza-Flavanones as potent cross-species microRNA inhibitors that arrest cell cycle

aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compounds were found to arrest cell cycle via a novel cross species microRNA-dependent regulatory pathway interpreting an unexpected link between cell cycle arrest and microRNA mediated control in cancer.     

Effects of growth factors on cell cycle arrest in dolichyl phosphate-depleted cultures

Previously we showed that CHO cell growth is arrested in the G1 or G0 phase within 24 h after the biosynthesis of mevalonic acid is blocked. The growth-limiting factor under these conditions appeared to be dolichyl phosphate or one of its glycosylated derivatives with consequent decrease in the synthesis of N-linked glycoproteins (Doyle, J.W., and A.A. Kandutsch, 1988, J. Cell Physiol. 137:133–140; Kabakoff, B., J.W. Doyle, and A.A. Kandutsch, 1990, Arch. Biochem. Biophys. 276:382–389). We show herein that cell surface glycoproteins are depleted in the inhibited cultures and that growth arrest is delayed when supraphysiological concentrations of insulin, insulin-like growth factor-1 (IGF-1) and bFGF are added to the culture medium. Apparently an elevated level of a growth factor increases the length of time during which a threshold level of occupied receptor is maintained as the number of glycosylated receptor molecules declines. The results support the idea that cellular levels of dolichyl phosphate and its derivatives may limit cell division by controlling the numbers of functional receptors for growth factors and of other glycoproteins on the cell surface. © 1993 Wiley-Liss, Inc.     

Phosphatidate phosphatase: activity and properties in fetal and adult rat lung.

The purpose of this work is to compare the properties of phosphatidate phosphatase (L-alpha-phosphatidate phosphohydrolase, EC 3.1.3.4) in fetal and adult rat lung and to establish the developmental profile of activity measured under optimal conditions. The maximal pH of 6.0--7.0 and the inhibition by fluoride, Ca2+ and detergents were simialr for both adult and fetal. Phosphatidate phosphohydrolase activity was located in both mitochondria and microsomes. The localizations of marker enzymes indicated that the activity in these subfractions was not a result of cross contaminations. Very low activity was detected in the supernatant fraction and no Mg2+ requirement was demonstrable. The activity in the particulate fraction was about 50% of the adult from 18 day gestation until birth. Following birth, the activity rapidly increased to adult levels. Dipalmitoyl, dioleoyl and diacyl glycerol 3-phosphates are all utilized well as substrates. 1,2-dipalmitoyl-sn-glycerol 3-phosphate was hydrolyzed faster under maximal conditions. The velocity-substrate curves tended to be sigmoidal, particularly when 1,2-dipalmitoyl-sn-glycerol 3-phosphate was the substrate. Estimated apparent Km values of 0.02--0.03 mM were obtained for fetal and adult preparations.     

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.     

A role for mesenchyme dynamics in mouse lung branching morphogenesis

Mammalian airways are highly ramified tree-like structures that develop by the repetitive branching of the lung epithelium into the surrounding mesenchyme through reciprocal interactions. Based on a morphometric analysis of the epithelial tree, it has been recently proposed that the complete branching scheme is specified early in each lineage by a programme using elementary patterning routines at specific sites and times in the developing lung. However, the coupled dynamics of both the epithelium and mesenchyme have been overlooked in this process. Using a qualitative and quantitative in vivo morphometric analysis of the E11.25 to E13.5 mouse whole right cranial lobe structure, we show that beyond the first generations, the branching stereotypy relaxes and both spatial and temporal variations are common. The branching pattern and branching rate are sensitive to the dynamic changes of the mesoderm shape that is in turn mainly dependent upon the volume and shape of the surrounding intrathoracic organs. Spatial and temporal variations of the tree architecture are related to local and subtle modifications of the mesoderm growth. Remarkably, buds never meet after suffering branching variations and continue to homogenously fill the opening spaces in the mesenchyme. Moreover despite inter-specimen variations, the growth of the epithelial tree and the mesenchyme remains highly correlated over time at the whole lobe level, implying a long-range regulation of the lung lobe morphogenesis. Together, these findings indicate that the lung epithelial tree is likely to adapt in real time to fill the available space in the mesenchyme, rather than being rigidly specified and predefined by a global programme. Our results strongly support the idea that a comprehensive understanding of lung branching mechanisms cannot be inferred from the branching pattern or behavior alone. Rather it needs to be elaborated upon with the reconsideration of mesenchyme-epithelium coupled growth and lung tissues mechanics.