Proteome changes of human bronchial epithelial cells in response to pro-inflammatory mediator leukotriene E4 and pro-remodelling factor TGF-β1

Inflammatory environment chronically activates bronchial epithelial cells to stimulate airway cells including epithelial cells themselves by secreting pro-inflammatory and regulatory factors. Proteomic approach is most relevant to screen the epithelial pathways following the inflammatory stimuli. We compared protein expression of the human bronchial epithelial cells exposed to leukotriene E₄ (LTE₄) and transforming growth factor-β₁ (TGF-β₁) with that of non-stimulated cells. The proteins were separated by 2-DE and the differentially expressed proteins were identified by MALDI-TOF MS and TOF/TOF tandem MS/MS. This approach allowed identification of 31 proteins, of which 26 corresponded to different proteins. β-tubulin, significantly down-regulated by LTE₄, was confirmed as a ciliated cell marker β-tubulin IV, whose decrease by LTE₄ was further corroborated by flow cytometry and RT-qPCR. This refers to a contribution of cysteinyl leukotrienes to epithelial remodelling and initiation of epithelial-mesenchymal transition in conducting airways. Of the affected proteins by TGF-β₁, clinically most relevant ones were up-regulated antioxidant enzyme superoxide dismutase 1, pro-fibrotic enzyme protein disulfide-isomerase and heat shock 70 kDa protein 9B. The changed protein profiles from this study add novel aspects to improve our understanding of the airway pathobiology, provide hints for further directed airway research and may contribute to selecting targets for future therapeutics.     

Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/β-catenin pathway in IPF. The potential impact of WNT/β-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/β-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16–24 months) versus young (3 months) mice had relatively less epithelial (EpCAM⁺) but more inflammatory (CD45⁺) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/β-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/β-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/β-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/β-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/β-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.     

Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.     

Constitutive activation of β-catenin in ameloblasts leads to incisor enamel hypomineralization

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/β-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/β-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of β-catenin in ameloblasts, we established Amelx-Cre; β-catenin^?ex3fl/fl (CA-β-catenin) mice, which could constitutive activate β-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-β-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-β-catenin mice. These data indicated that β-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.     

Distribution of E-cadherin and β-catenin in relation to cell maturation and cell extrusion in rat and mouse small intestines

In the small intestines, cell renewal from stem cells present in the crypts is balanced by cell extrusion from the tips of the villi. The mechanism by which extrusion occurs is unknown. Recent in vitro data suggested that loss of E-cadherin could contribute to cell extrusion and induction of programmed cell death (PCD) in mouse small intestinal epithelium. We have studied if this also occurs in the intact rodent small intestine. Our results confirm that extruded cells are negative for E-cadherin. However, loss of the E-cadherin-interacting protein β-catenin preceded both extrusion and loss of E-cadherin. Thus, all extruded cells as well as all cells in the process of extrusion lacked staining for β-catenin. Moreover, almost 80% of all cells undergoing programmed cell death, as detected by the TUNEL reaction, lacked β-catenin whereas over 70% of such cells were positive for E-cadherin. However, most cells lacking β-catenin did not display signs of PCD as detected by the TUNEL method or by staining for active caspase-3. Therefore, these results suggest that loss of β-catenin precedes the onset of programmed cell death, loss of E-cadherin and extrusion from the villi.     

Mitogenic response of rat lung and tracheal epithelial cells in monolayer primary cultures—Modulation of TNF-α expression

Epithelial cells isolated from rat lung and trachea were grown on monolayers and their response to a number of hormones and growth factors were studied. Maximum proliferative response in serum containing media was observed when insulin, cholera toxin and cortisol were present together. However, these additives when present independently showed a marginal response. The synergism, due to these factors in promoting growth was seen very early in culture (day 4) as shown by thymidine labelling studies, On examining the indices of early mitogenesis, such as the expression ofc-myc, our data suggests that these factors stimulate the expression ofc-myc within 4 h. With respect to expression of TNF-α mRNA, this study suggests a possible modulation of TNF-α expression in response to these mitogens that stimulate proliferation maximally. Whether this expression of TNF-α by these epithelial cells is due to a maximal proliferative stimulus and/or is an early step in the cascade of intracellular signalling events is to be investigated in detail.     

Spindle checkpoint and apoptotic response in α-particle transformed human bronchial epithelial cells

The mitotic spindle checkpoint and apoptosis in response to nocodazole, a microtubule-disrupting agent, were investigated in the -particle transformed human bronchial epithelial cell lines BERP35T1, BERP35T4 and the parental BEP2D cell line. When treated with 0.2 g/ml of nocodazole, BEP2D and BERP35T1 cells were efficiently arrested in the mitotic phase, whilst BERP35T4, a transformed cell line showing chromosomal instability, failed to be arrested as evidenced by a low G2/M fraction. BERP35T4 cells also showed a higher proportion of aneuploids when treated with nocodazole or not. Thus, the BERP35T4 cell line has a defect in spindle checkpoint function. The extent of apoptosis induced by nocodazole (0.3 g/ml) was significantly higher (2-fold to 2.5-fold) in BEP2D cells than in the two transformed cell lines. Furthermore, the induced apoptosis was found to occur predominantly before mitotic division in BEP2D cells. In BERP35T4 cells, however, 50% of induced apoptosis occurred before mitotic division and 50% occurred after division in binucleated cells when co-treated with cytochalasin B. The 5-CpG island of the Chfr gene, a mitotic checkpoint gene that functions in entry into metaphase, was found to be methylated in BERP35T4 cells but not in BEP2D cells. Consistent with methylation, the expression of the Chfr gene was markedly suppressed in BERP35T4 cells. Our results suggest that the impaired spindle checkpoint and abnormal apoptotic response may be related to the oncogenic progression of human bronchial epithelial cells initiated by exposure to -particles.     

Human induced pluripotent stem cell–derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury

Background aims

Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD.

Loss of cadherin-binding proteins β-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis

Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.     

Absolute β-catenin concentrations in Wnt pathway-stimulated and non-stimulated cells

The intracellular level of the proto-oncoprotein β-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the β-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining β-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high β-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. β-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The β-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the β-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of β-catenin in colorectal tumours revealed high β-catenin levels in tumours with truncating mutations in the APC gene.     

The role of pleiotrophin and β-catenin in fetal lung development

Mammalian lung development is a complex biological process, which is temporally and spatially regulated by growth factors, hormones, and extracellular matrix proteins. Abnormal changes of these molecules often lead to impaired lung development, and thus pulmonary diseases. Epithelial-mesenchymal interactions are crucial for fetal lung development. This paper reviews two interconnected pathways, pleiotrophin and Wnt/β-catenin, which are involved in fibroblast and epithelial cell communication during fetal lung development.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

Metformin promotes Mycobacterium tuberculosis killing and increases the production of human β-defensins in lung epithelial cells and macrophages

Diabetes has been associated with an increased risk of developing tuberculosis. The reasons related to the increased susceptibility to develop TB in type 2 diabetes mellitus (T2DM) individuals, has not been completely elucidated. However, this susceptibility has been attributed to several factors including failures and misfunctioning of the immune system. In the present study, we aimed to determine the role of anti-hyperglycemic drugs such as glyburide, insulin, and metformin to promote the killing of mycobacteria through the regulation of innate immune molecules such as host defense peptides (HDP) in lung epithelial cells and macrophages. Our results showed that metformin reduces bacillary loads in macrophages and lung epithelial cells which correlates with higher production of β-defensin-2, -3 and -4. Since β-defensins are crucial molecules for controlling Mycobacterium tuberculosis growth, the present results suggest that the use of metformin would be the first choice in the treatment for T2DM2, in patients within tuberculosis-endemic areas.