Domokos Bartis

Epithelial–mesenchymal transition in lung development and disease: does it exist and is it important?

Epithelial–mesenchymal transition (EMT) is a process when epithelial cells gradually transform into mesenchymal-like cells losing their epithelial functionality and characteristics. EMT is thought to be involved in the pathogenesis of numerous lung diseases ranging from developmental disorders, fibrotic tissue remodelling to lung cancer. The most important question—namely what is the importance and contribution of EMT in the pathogenesis of several chronic lung conditions (asthma, COPD, bronchiolitis obliterans syndrome and lung fibrosis)—is currently intensely debated. This review gives a brief insight into the mechanism and assessment methods of EMT in various pulmonary diseases and summarises the recent literature highlighting the controversial experimental data and conclusions.

Emerging pathways of non-genomic glucocorticoid (GC) signalling in T cells

In the last decade new glucocorticoid (GC)-signalling mechanisms have emerged. The evolving field of non-genomic GC actions was precipitated from two major directions: (i) some rapid/acute clinical GC applications could not be explained based on the relatively slowly appearing genomic GC action and (ii) accumulating evidence came to light about the discrepancy in the apoptosis sensitivity and GR expression of thymocytes and other lymphoid cell types. Herein, we attempt to sample the latest information in the field of non-genomic GC signalling in T cells, and correlate it with results from our laboratory. We discuss some aspects of the regulation of thymocyte apoptosis by GCs, paying special interest to the potential role(s) of mitochondrial GR signalling. The interplay between the T cell receptor (TcR) and glucocorticoid receptor (GR) signalling pathways is described in more detail, focusing on ZAP-70, which is a novel target of rapid GC action.

Wnt4 and LAP2alpha as Pacemakers of Thymic Epithelial Senescence

Age-associated thymic involution has considerable physiological impact by inhibiting de novo T-cell selection. This impaired T-cell production leads to weakened immune responses. Yet the molecular mechanisms of thymic stromal adipose involution are not clear. Age-related alterations also occur in the murine thymus providing an excellent model system. In the present work structural and molecular changes of the murine thymic stroma were investigated during aging. We show that thymic epithelial senescence correlates with significant destruction of epithelial network followed by adipose involution. We also show in purified thymic epithelial cells the age-related down-regulation of Wnt4 (and subsequently FoxN1), and the prominent increase in LAP2α expression. These senescence-related changes of gene expression are strikingly similar to those observed during mesenchymal to pre-adipocyte differentiation of fibroblast cells suggesting similar molecular background in epithelial cells. For molecular level proof-of-principle stable LAP2α and Wnt4-over-expressing thymic epithelial cell lines were established. LAP2α over-expression provoked a surge of PPARγ expression, a transcription factor expressed in pre-adipocytes. In contrast, additional Wnt4 decreased the mRNA level of ADRP, a target gene of PPARγ. Murine embryonic thymic lobes have also been transfected with LAP2α- or Wnt4-encoding lentiviral vectors. As expected LAP2α over-expression increased, while additional Wnt4 secretion suppressed PPARγ expression. Based on these pioneer experiments we propose that decreased Wnt activity and increased LAP2α expression provide the molecular basis during thymic senescence. We suggest that these molecular changes trigger thymic epithelial senescence accompanied by adipose involution. This process may either occur directly where epithelium can trans-differentiate into pre-adipocytes; or indirectly where first epithelial to mesenchymal transition (EMT) occurs followed by subsequent pre-adipocyte differentiation. The latter version fits better with literature data and is supported by the observed histological and molecular level changes.

Mitochondrial translocation of the glucocorticoid receptor in double-positive thymocytes correlates with their sensitivity to glucocorticoid-induced apoptosis

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.

Down-regulation of canonical and up-regulation of non-canonical Wnt signalling in the carcinogenic process of squamous cell lung carcinoma.

The majority of lung cancers (LC) belong to the non-small cell lung carcinoma (NSCLC) type. The two main NSCLC sub-types, namely adenocarcinoma (AC) and squamous cell carcinoma (SCC), respond differently to therapy. Whereas the link between cigarette smoke and lung cancer risk is well established, the relevance of non-canonical Wnt pathway up-regulation detected in SCC remains poorly understood. The present study was undertaken to investigate further the molecular events in canonical and non-canonical Wnt signalling during SCC development. A total of 20 SCC and AC samples with matched non-cancerous controls were obtained after surgery. TaqMan array analysis confirmed up-regulation of non-canonical Wnt5a and Wnt11 and identified down-regulation of canonical Wnt signalling in SCC samples. The molecular changes were tested in primary small airway epithelial cells (SAEC) and various lung cancer cell lines (e.g. A549, H157, etc). Our studies identified Wnt11 and Wnt5a as regulators of cadherin expression and potentiated relocation of β-catenin to the nucleus as an important step in decreased cellular adhesion. The presented data identifies additional details in the regulation of SCC that can aid identification of therapeutic drug targets in the future.

Vitamin D deficiency contributes directly to the acute respiratory distress syndrome (ARDS)

Rationale Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and intensive therapy unit mortality but has not been assessed as a risk factor for acute respiratory distress syndrome (ARDS). Causality of these associations has never been demonstrated. Objectives To determine if ARDS is associated with vitamin D deficiency in a clinical setting and to determine if vitamin D deficiency in experimental models of ARDS influences its severity. Methods Human, murine and in vitro primary alveolar epithelial cell work were included in this study. Findings Vitamin D deficiency (plasma 25(OH)D levels <50 nmol/L) was ubiquitous in patients with ARDS and present in the vast majority of patients at risk of developing ARDS following oesophagectomy. In a murine model of intratracheal lipopolysaccharide challenge, dietary-induced vitamin D deficiency resulted in exaggerated alveolar inflammation, epithelial damage and hypoxia. In vitro, vitamin D has trophic effects on primary human alveolar epithelial cells affecting >600 genes. In a clinical setting, pharmacological repletion of vitamin D prior to oesophagectomy reduced the observed changes of in vivo measurements of alveolar capillary damage seen in deficient patients. Conclusions Vitamin D deficiency is common in people who develop ARDS. This deficiency of vitamin D appears to contribute to the development of the condition, and approaches to correct vitamin D deficiency in patients at risk of ARDS should be developed. Trial registration UKCRN ID 11994.

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.

Multiple suppression pathways of canonical Wnt signalling control thymic epithelial senescence

Highlights ► Active Wnt-4 receptors, Fz-4 and Fz-6 are up-regulated during initialisation of thymic atrophy. ► Inhibitory receptor Fz-6, a negative regulator of β-catenin dependent Wnt signalling is increasingly associated with PKCδ during initialisation of thymic ageing. ► One of the Wnt-4 target genes is connective tissue growth factor (CTGF). ► CTGF, a suppressor of canonical Wnt signalling is up-regulated during ageing. ► Decline of the thymic epithelial network is initiated by active suppression of Wnt signalling.

Developmental shift in TcR-mediated rescue of thymocytes from glucocorticoid-induced apoptosis

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.

Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence

In the aging lung, the lung capacity decreases even in the absence of diseases. The progenitor cells of the distal lung, the alveolar type II cells (ATII), are essential for the repair of the gas‐exchange surface. Surfactant protein production and survival of ATII cells are supported by lipofibroblasts that are peroxisome proliferator‐activated receptor gamma (PPARγ)‐dependent special cell type of the pulmonary tissue. PPARγ levels are directly regulated by Wnt molecules; therefore, changes in the Wnt microenvironment have close control over maintenance of the distal lung. The pulmonary aging process is associated with airspace enlargement, decrease in the distal epithelial cell compartment and infiltration of inflammatory cells. qRT–PCR analysis of purified epithelial and nonepithelial cells revealed that lipofibroblast differentiation marker parathyroid hormone‐related protein receptor (PTHrPR) and PPARγ are reduced and that PPARγ reduction is regulated by Wnt4 via a β‐catenin‐dependent mechanism. Using a human in vitro 3D lung tissue model, a link was established between increased PPARγ and pro‐surfactant protein C (pro‐SPC) expression in pulmonary epithelial cells. In the senile lung, both Wnt4 and Wnt5a levels increase and both Wnt‐s increase myofibroblast‐like differentiation. Alteration of the Wnt microenvironment plays a significant role in pulmonary aging. Diminished lipo‐ and increased myofibroblast‐like differentiation are directly regulated by specific Wnt‐s, which process also controls surfactant production and pulmonary repair mechanisms.