LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1+/−mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5LGL1KD). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5LGL1KDsuppressed cell growth and increased apoptosis of annexin V+staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5LGL1KDcells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

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Trop2 regulates motility and lamellipodia formation in cultured fetal lung fibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00160.2012 ◽

2013 ◽

Vol 305 (7) ◽

pp. L508-L521 ◽

Cited By ~ 8

Author(s):

Annie R. A. McDougall ◽

Stuart B. Hooper ◽

Valerie A. Zahra ◽

Timothy J. Cole ◽

Camden Y. Lo ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Migration ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Mrna Levels ◽

Fetal Lung Development ◽

Altered Cell ◽

Critical Components ◽

And Migration

Proliferation and migration of fibroblasts are vital for fetal lung development. However, the regulatory mechanisms are poorly understood. We have previously shown that TROP2 gene expression is closely associated with fetal lung cell proliferation in vivo and that TROP2 knockdown decreases proliferation of fetal lung fibroblasts in culture. We hypothesized that the Trop2 protein also regulates the morphology and motility of fetal lung fibroblasts. Fibroblasts isolated from fetal rat lungs (gestational age embryonic day 19) adopted a myofibroblast-like morphology in culture. Trop2 protein was localized to lamellipodia. TROP2 siRNA significantly decreased: TROP2 mRNA levels by 77%, the proportion of cells containing Trop2 protein by 70%, and cell proliferation by 50%. TROP2 siRNA also decreased the degree of motility as determined by the number of gridlines that cells moved across (2.2 ± 0.2 vs. 3.2 ± 0.2; P < 0.001). TROP2 knockdown altered cell morphology, causing a notable absence of lamellipodia and abnormal localization of components of the cell migration apparatus, and it reduced phosphorylated ERK1 and ERK2 levels. In contrast, TROP2 overexpression significantly increased: TROP2 mRNA levels by 40-fold, cell proliferation by 40%, the proportion of cells that were motile by 20%, and the number of gridlines that cells moved across (2.1 ± 0.2 vs. 1.6 ± 0.1; P < 0.001). Our data suggest that Trop2 regulates cell proliferation and motility and that it does so by regulating the ERK pathway and several critical components of the cell migration apparatus.

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Collagen secretion mediated by NMDR activation in human fetal lung fibroblasts

Cell Biology International ◽

10.1042/cbi034s070a ◽

2010 ◽

Vol 34 (8) ◽

pp. S70-S70

Author(s):

MingJie WANG ◽

ZiQiang LUO ◽

Mei LU ◽

LiHong SHANG ◽

ShaoJie YUE

Keyword(s):

Fetal Lung ◽

Lung Fibroblasts ◽

Collagen Secretion ◽

Human Fetal Lung

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Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l227 ◽

1997 ◽

Vol 273 (1) ◽

pp. L227-L233 ◽

Cited By ~ 23

Author(s):

V. C. Venkatesh ◽

H. D. Katzberg

Keyword(s):

Rna Polymerase Ii ◽

Actinomycin D ◽

Critical Role ◽

Fetal Lung ◽

Explant Culture ◽

Late Gestation ◽

Na Channel ◽

Second Trimester ◽

Cyclic Monophosphate ◽

Human Fetal Lung

Pulmonary epithelial Na+ channels (ENaC), composed of three distinct subunits (alpha, beta, and gamma), play a critical role in the regulation of fluid reabsorption from airspaces of late-gestation fetal lung. We studied the expression of ENaC subunit genes in cultured human fetal lung. All three mRNAs were expressed at low levels in second trimester lung (13-32% of adult values at 24 wk gestation). There was a spontaneous increase of approximately threefold over preculture values of all three subunits within 24 h of explant culture in serum-free Waymouth's medium. Dexamethasone (Dex) induced all three mRNAs by two- to threefold. Maximal induction was noted by 8 h with 30-100 nM Dex and half-maximal stimulation with 3-10 nM Dex. Cycloheximide decreased basal expression of all three subunits by 8 h but did not alter the response to Dex. Actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), inhibitors of RNA polymerase II, decreased the basal and the Dex-induced expression of all three subunits with a more marked effect on human hENaC-gamma than on hENaC-alpha or hENaC-beta. Under conditions where transcription was blocked by actinomycin D or DRB, Dex did not alter the stability of the three mRNAs. Triiodothyronine (T3) at low (2 nM) or high (100 nM) concentrations had no effect on the expression of the three subunits in the presence or absence of low (10 nM) or high (100 nM) concentrations of Dex for 8 or 24 h. Similarly, 8-bromoadenosine 3',5'-cyclic monophosphate (2 microM) had no effect on basal or Dex-induced increase in the three subunits. We conclude that the three Na+ channel subunit genes are expressed in second trimester human fetal lung and are coordinately upregulated by glucocorticoid hormones but not by T3 or adenosine 3',5'-cyclic monophosphate. Glucocorticoid induction is receptor mediated, is primarily transcriptional, and does not require the induction of an intermediate protein for transcriptional enhancement. We speculate that induction of lung ENaC may contribute to the beneficial effects of antenatal glucocorticoids in premature babies.

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MORPHOGENETIC ASPECTS OF MULTILAYERING IN PETRI DISH CULTURES OF HUMAN FETAL LUNG FIBROBLASTS

The Journal of Cell Biology ◽

10.1083/jcb.41.1.298 ◽

1969 ◽

Vol 41 (1) ◽

pp. 298-311 ◽

Cited By ~ 42

Author(s):

Tom Elsdale ◽

Robert Foley

Keyword(s):

Extracellular Matrix ◽

Human Lung ◽

Petri Dish ◽

Fetal Lung ◽

Single Species ◽

Lung Fibroblasts ◽

Human Lung Fibroblasts ◽

Spontaneous Aggregation ◽

Control Hypothesis ◽

Human Fetal Lung

Randomly seeded Petri dish cultures of embryonic human lung fibroblasts generate, in the course of their growth, highly ordered cellular arrangements. Thick, bilaterally symmetrical ridges with an axial polarity and an orthogonal, multilayered internal organization are observed within stationary cultures. The generation of these structures has been investigated. Ridges result from the spontaneous aggregation of cells in postconfluent cultures brought about by directed cell movements. These movements are promoted by the localized production of extracellular matrix sheets containing collagen, which provide new substrates for cellular colonization. Cells that have colonized one matrix substrate may secrete another above themselves, which will in turn be colonized. By a continuation of this cycle, thick stacks consisting of alternate layers of cells and matrix are produced to yield the observed aggregations. The distribution and shape of ridges in a culture imply that matrix substrates are confined to specific locations. The suggested control hypothesis assumes that all the cells in fibroblast cultures are potential producers of a single species of matrix. The serviceability of this matrix as a substrate for cellular colonization, however, is destroyed if the producer cells are motile. Matrix substrates, therefore, are only made by nonmotile cells.

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Prostacyclin analogs inhibit fibroblast migration

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00432.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. L428-L432 ◽

Cited By ~ 25

Author(s):

Tadashi Kohyama ◽

Xiangde Liu ◽

Hui Jung Kim ◽

Tetsu Kobayashi ◽

Ronald F. Ertl ◽

...

Keyword(s):

Tissue Repair ◽

Fetal Lung ◽

Inhibitory Effect ◽

Lung Fibroblasts ◽

Fibroblast Migration ◽

Inflammatory Processes ◽

Dependent Protein ◽

Pka Pathway ◽

Human Fetal Lung ◽

Abnormal Tissue

The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI2) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI2on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI2analog carbaprostacyclin (10−6M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 μg/ml) 58.0 ± 13.2% ( P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 ± 4.6% ( P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI2analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10−6M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI2appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.

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Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(81)90235-6 ◽

1981 ◽

Vol 210 (2) ◽

pp. 678-690 ◽

Cited By ~ 8

Author(s):

Hilda H. Carnicero ◽

Anthony M. Adamany ◽

Sasha Englard

Keyword(s):

Fetal Lung ◽

Lung Fibroblasts ◽

Human Fetal Lung

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Loss of FLCN inhibits canonical WNT signaling via TFE3

Human Molecular Genetics ◽

10.1093/hmg/ddz158 ◽

2019 ◽

Vol 28 (19) ◽

pp. 3270-3281 ◽

Cited By ~ 8

Author(s):

John C Kennedy ◽

Damir Khabibullin ◽

Thomas Hougard ◽

Julie Nijmeh ◽

Wei Shi ◽

...

Keyword(s):

Wnt Pathway ◽

Lower Lobe ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Pulmonary Cysts ◽

Wnt Ligands ◽

Neonatal Lung ◽

Canonical Wnt ◽

Human Fetal Lung ◽

Bhd Syndrome

Abstract Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt–Hogg–Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/β inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/β inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression—two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/β inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/β inhibition. Finally, we found that a constitutively active β-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

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