Oxidative stress and NF-κB signaling are involved in LPS induced pulmonary dysplasia in chick embryos

Inflammation or dysbacteriosis-derived lipopolysaccharides (LPS) adversely influence the embryonic development of respiratory system. However, the precise pathological mechanisms still remain to be elucidated. In this study, we demonstrated that LPS exposure caused lung maldevelopment in chick embryos, including higher embryo mortality, increased thickness of alveolar gas exchange zone, and accumulation of PAS⁺ immature pulmonary cells, accompanied with reduced expression of alveolar epithelial cell markers and lamellar body count. Upon LPS exposure, pulmonary cell proliferation was significantly altered and cell apoptosis was inhibited as well, indicating a delayed progress of pulmonary development. LPS treatment also resulted in reduced CAV-1 expression and up-regulation of Collagen I, suggesting increased lung fibrosis, which was verified by Masson staining. Moreover, LPS induced enhanced Nrf2 expression in E18 lungs, and the increased reactive oxygen species (ROS) production was confirmed in MLE-12 cells in vitro. Antioxidant vitamin C restored the LPS induced down-regulation of ABCA3, SP-C and GATA-6 in MLE-12 cells. Furthermore, LPS induced activation of NF-κB signaling in MLE-12 cells, and the LPS-induced decrease in SP-C expression was partially abrogated by blocking NF-κB signaling with Bay-11–7082. Bay-11–7082 also inhibited LPS-induced increases of ROS and Nrf2 expression. Taken together, we have demonstrated that oxidative stress and NF-κB signaling are involved in LPS induced disruption of pulmonary cell development in chick embryos.     

Alk5-mediated transforming growth factor β signaling acts upstream of fibroblast growth factor 10 to regulate the proliferation and maintenance of dental epithelial stem cells

Mouse incisors grow continuously throughout life. This growth is supported by the division of dental epithelial stem cells that reside in the cervical loop region. Little is known about the maintenance and regulatory mechanisms of dental epithelial stem cells. In the present study, we investigated how transforming growth factor β (TGF-β) signaling-mediated mesenchymal-epithelial cell interactions control dental epithelial stem cells. We designed two approaches using incisor organ culture and bromodeoxyuridine (BrdU) pulse-chase experiments to identify and evaluate stem cell functions. We show that the loss of the TGF-β type I receptor (Alk5) in the cranial neural crest-derived dental mesenchyme severely affects the proliferation of TA (transit-amplifying) cells and the maintenance of dental epithelial stem cells. Incisors of Wnt1-Cre; Alk5(fl/fl) mice lost their ability to continue to grow in vitro. The number of BrdU label-retaining cells (LRCs) was dramatically reduced in Alk5 mutant mice. Fgf10, Fgf3, and Fgf9 signals in the dental mesenchyme were downregulated in Wnt1-Cre; Alk5(fl/fl) incisors. Strikingly, the addition of exogenous fibroblast growth factor 10 (FGF10) into cultured incisors rescued dental epithelial stem cells in Wnt1-Cre; Alk5(fl/fl) mice. Therefore, we propose that Alk5 functions upstream of Fgf10 to regulate TA cell proliferation and stem cell maintenance and that this signaling mechanism is crucial for stem cell-mediated tooth regeneration.     

Activation of Wnt signalling reduces the population of cancer stem cells in ameloblastoma

ObjectivesThe treatment of ameloblastoma, an odontogenic epithelial tumour destroying jawbone, mainly depends on radical destructive resections. Other therapeutic options are limited by the characteristics of ameloblastoma, such as high recurrence rates and resistance to radiation and chemotherapy, which implies possible existence of cancer stem cells (CSCs) in ameloblastoma. Here, we identified a putative CSC population in immortalized and primary human ameloblastoma cells and examined possible therapeutic reagents to reduce the CSC population.MethodsWe investigated subpopulations of AM‐1 cell line and human ameloblastoma cells using immunocytochemistry and flow cytometry and the effects of Wnt signalling activators on the 2‐ and 3‐dimensional cultured ameloblastoma cells using molecular biological analyses.ResultAmong heterogenous ameloblastoma cells, small‐sized and round‐shaped cells were found to be proliferative and expressed a marker of dental epithelial stem cells, SRY‐box 2 (Sox2). Exogenous activation of Wnt signalling using glycogen synthase kinase 3β inhibitors, lithium chloride (LiCl) and valproic acid (VPA), increased the cell size and decreased proliferation of cells and expression of Sox2 in 2 dimensionally cultured AM‐1 and human primary ameloblastoma cells. Furthermore, the growth of 3 dimensionally cultured AM‐1 cells as suspended or embedded in gel was suppressed by treatment with Wnt signalling activators, VPA and CHIR99021, or antibodies to sclerostin, an antagonist of Wnt signalling.ConclusionWe suggest that Wnt signalling activators are potential drug candidates to suppress CSCs in ameloblastoma.     

c-Myc Regulates Proliferation and Fgf10 Expression in Airway Smooth Muscle after Airway Epithelial Injury in Mouse

During lung development, Fibroblast growth factor 10 (Fgf10), which is expressed in the distal mesenchyme and regulated by Wnt signaling, acts on the distal epithelial progenitors to maintain them and prevent them from differentiating into proximal (airway) epithelial cells. Fgf10-expressing cells in the distal mesenchyme are progenitors for parabronchial smooth muscle cells (PSMCs). After naphthalene, ozone or bleomycin-induced airway epithelial injury, surviving epithelial cells secrete Wnt7b which then activates the PSMC niche to induce Fgf10 expression. This Fgf10 secreted by the niche then acts on a subset of Clara stem cells to break quiescence, induce proliferation and initiate epithelial repair. Here we show that conditional deletion of the Wnt target gene c-Myc from the lung mesenchyme during development does not affect proper epithelial or mesenchymal differentiation. However, in the adult lung we show that after naphthalene-mediated airway epithelial injury c-Myc is important for the activation of the PSMC niche and as such induces proliferation and Fgf10 expression in PSMCs. Our data indicate that conditional deletion of c-Myc from PSMCs inhibits airway epithelial repair, whereas c-Myc ablation from Clara cells has no effect on airway epithelial regeneration. These findings may have important implications for understanding the misregulation of lung repair in asthma and COPD.     

Hoxa-5 in mouse developing lung: cell-specific expression and retinoic acid regulation

Hoxa-5 is a homeobox gene that is highly expressed in the developing mouse lung. However, little is known about the molecular mechanisms controlling expression. We characterized the ontogeny of Hoxa-5 gene and protein expressions during lung development and then studied the cell-specific effects of retinoic acid (RA) on Hoxa-5 mRNA in fetal lung fibroblasts and MLE-12 mouse lung epithelial cells. Strong but constant Hoxa-5 gene and protein expressions were detected from mouse lung on embryonic day 13.5 to postnatal day 2. At baseline, the gene was strongly expressed in the fibroblasts of day 17.5 fetal mouse lungs. A very weak but reproducible expression was present in the MLE-12 cells. RA stimulated gene expression in both cell types in a time- and dose-dependent manner. Peak expression occurred much later in the MLE-12 cells compared with that in fibroblasts. Cycloheximide and actinomycin D treatment studies suggested that the differences in RA effect on each cell type may involve the presence of a repressor that can be overcome by RA.     

Sprouty proteins regulate ureteric branching by coordinating reciprocal epithelial Wnt11, mesenchymal Gdnf and stromal Fgf7 signalling during kidney development

The kidney is a classic model for studying mechanisms of inductive tissue interactions associated with the epithelial branching common to many embryonic organs, but the molecular mechanisms are still poorly known. Sprouty proteins antagonize tyrosine kinases in the Egf and Fgf receptors and are candidate components of inductive signalling in the kidney as well. We have addressed the function of sprouty proteins in vivo by targeted expression of human sprouty 2 (SPRY2) in the ureteric bud, which normally expresses inductive signals and mouse sprouty 2 (Spry2). Ectopic SPRY2 expression led to postnatal death resulting from kidney failure, manifested as unilateral agenesis, lobularization of the organ or reduction in organ size because of inhibition of ureteric branching. The experimentally induced dysmorphology associated with deregulated expression of Wnt11, Gdnf and Fgf7 genes in the early stages of organogenesis indicated a crucial role for sprouty function in coordination of epithelial-mesenchymal and stromal signalling, the sites of expression of these genes. Moreover, Fgf7 induced Spry2 gene expression in vitro and led with Gdnf to a partial rescue of the SPRY2-mediated defect in ureteric branching. Remarkably, it also led to supernumerary epithelial bud formation from the Wolffian duct. Together, these data suggest that Spry genes contribute to reciprocal epithelial-mesenchymal and stromal signalling controlling ureteric branching, which involves the coordination of Ffg/Wnt11/Gdnf pathways.     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

IL‐17A‐producing T cells exacerbate fine particulate matter‐induced lung inflammation and fibrosis by inhibiting PI3K/Akt/mTOR‐mediated autophagy

Fine particulate matter (PM2.5) is the primary air pollutant that is able to induce airway injury. Compelling evidence has shown the involvement of IL‐17A in lung injury, while its contribution to PM2.5‐induced lung injury remains largely unknown. Here, we probed into the possible role of IL‐17A in mouse models of PM2.5‐induced lung injury. Mice were instilled with PM2.5 to construct a lung injury model. Flow cytometry was carried out to isolate γδT and Th17 cells. ELISA was adopted to detect the expression of inflammatory factors in the supernatant of lavage fluid. Primary bronchial epithelial cells (mBECs) were extracted, and the expression of TGF signalling pathway‐, autophagy‐ and PI3K/Akt/mTOR signalling pathway‐related proteins in mBECs was detected by immunofluorescence assay and Western blot analysis. The mitochondrial function was also evaluated. PM2.5 aggravated the inflammatory response through enhancing the secretion of IL‐17A by γδT/Th17 cells. Meanwhile, PM2.5 activated the TGF signalling pathway and induced EMT progression in bronchial epithelial cells, thereby contributing to pulmonary fibrosis. Besides, PM2.5 suppressed autophagy of bronchial epithelial cells by up‐regulating IL‐17A, which in turn activated the PI3K/Akt/mTOR signalling pathway. Furthermore, IL‐17A impaired the energy metabolism of airway epithelial cells in the PM2.5‐induced models. This study suggested that PM2.5 could inhibit autophagy of bronchial epithelial cells and promote pulmonary inflammation and fibrosis by inducing the secretion of IL‐17A in γδT and Th17 cells and regulating the PI3K/Akt/mTOR signalling pathway.     

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.     

Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.     

Understanding the role of growth factors in embryonic development: insights from the lens

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-β and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/β-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study.     

CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

Pirfenidone modulates macrophage polarization and ameliorates radiation-induced lung fibrosis by inhibiting the TGF-β1/Smad3 pathway

Radiation-induced lung injury (RILI) mainly contributes to the complications of thoracic radiotherapy. RILI can be divided into radiation pneumonia (RP) and radiation-induced lung fibrosis (RILF). Once RILF occurs, patients will eventually develop irreversible respiratory failure; thus, a new treatment strategy to prevent RILI is urgently needed. This study explored the therapeutic effect of pirfenidone (PFD), a Food and Drug Administration (FDA)-approved drug for (IPF) treatment, and its mechanism in the treatment of RILF. In vivo, C57BL/6 mice received a 50 Gy dose of X-ray radiation to the whole thorax with or without the administration of PFD. Collagen deposition and fibrosis in the lung were reversed by PFD treatment, which was associated with reduced M2 macrophage infiltration and inhibition of the transforming growth factor-β1 (TGF-β1)/Drosophila mothers against the decapentaplegic 3 (Smad3) signalling pathway. Moreover, PFD treatment decreased the radiation-induced expression of TGF-β1 and phosphorylation of Smad3 in alveolar epithelial cells (AECs) and vascular endothelial cells (VECs). Furthermore, IL-4–induced M2 macrophage polarization and IL-13–induced M2 macrophage polarization were suppressed by PFD treatment in vitro, resulting in reductions in the release of arginase-1 (ARG-1), chitinase 3-like 3 (YM-1) and TGF-β1. Notably, the PFD-induced inhibitory effects on M2 macrophage polarization were associated with downregulation of nuclear factor kappa-B (NF-κB) p50 activity. Additionally, PFD could significantly inhibit ionizing radiation-induced chemokine secretion in MLE-12 cells and consequently impair the migration of RAW264.7 cells. PFD could also eliminate TGF-β1 from M2 macrophages by attenuating the activation of TGF-β1/Smad3. In conclusion, PFD is a potential therapeutic agent to ameliorate fibrosis in RILF by reducing M2 macrophage infiltration and inhibiting the activation of TGF-β1/Smad3.     

Roles of Wnt8a during formation and patterning of the mouse inner ear

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle.