Judit E. Pongracz

Wnt signalling in lung development and diseases

There are several signalling pathways involved in lung organogenesis including Notch, TGFβ /BMP, Sonic hedgehog (Shh), FGF, EGF, and Wnt. Despite the widely acknowledged significance of Wnt signalling in embryonic lung development, the role of different Wnt pathways in lung pathologies has been slow to emerge.In this review, we will present a synopsis of current Wnt research with particular attention paid to the role of Wnt signals in lung development and in pulmonary diseases.

Establishment and functioning of intrathymic microenvironments

Summary:  The thymus supports the production of self‐tolerant T cells from immature precursors. Studying the mechanisms regulating the establishment and maintenance of stromal microenvironments within the thymus therefore is essential to our understanding of T‐cell production and ultimately immune system functioning. Despite our ability to phenotypically define stromal cell compartments of the thymus, the mechanisms regulating their development and the ways by which they influence T‐cell precursors are still unclear. Here, we review recent findings and highlight unresolved issues relating to the development and functioning of thymic stromal cells.

Thymic epithelial cells provide Wnt signals to developing thymocytes

Interactions with thymic stromal cells are known to be critical for the development of T cells from progenitors entering the thymus, yet the molecular mechanisms of stromal cell function remain poorly understood. Accumulating evidence has highlighted the importance of β‐catenin‐mediated activation of T cell factor (TCF)/lymphoid enhancer factor (LEF) transcription during thymocyte development. As regulation of this signaling pathway is controlled by binding of soluble Wnt proteins to cell surface Frizzled (Fz) receptors, we studied components of Wnt/Fz‐mediated signaling in thecontext of stromal cell regulation of thymocyte development. We show that mRNA for a variety of Wnt family members, notably Wnt‐4, Wnt‐7a and 7b, and Wnt‐10a and 10b, are expressed by thymic epithelium rather then by thymocytes, while thymocytes demonstrate a developmentally regulated pattern of Fz receptor expression. Collectively these findings suggest (1) a functional role for Wnt‐producing thymic epithelium in determining TCF/LEF‐mediated transcriptional regulation in Fz‐bearing thymocytes, and (2) a role for defined Wnt‐Fz interactions at successive stages of thymocyte maturation. In support of this we show that separation of thymocytes from Wnt‐producing epithelial cells and the thymic microenvironment, triggers β‐catenin phosphorylation and degradation in thymocytes. Thus, sustained exposure to Wnt in the context of an intact stromal microenvironment is necessary for stabilization of β‐catenin‐mediated signaling in thymocytes.

Notch ligand‐bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling

Thymic epithelial cells are specialized to play essential roles at multiple stages of T cell development in the thymus, yet the molecular basis of this specialization is largely unknown. Recently, the Notch family of transmembrane proteins has been implicated in thymocyte development. Such proteins interact with cell surface proteins of the Delta‐like and Jagged families. It is known that Notch ligands are expressed intrathymically, and that Notch signaling is regulated by Notch ligands expressed on either the same or third‐party cells. However, functional analysis of Notch ligand expression, and elucidation of the mechanism of Notch ligand signaling in thymocyte development, are unclear. Here, we find that Notch ligand expression in the thymus is compartmentalized, with MHC class II+ thymic epithelium, but not thymocytes nor dendritic cells, expressing Jagged‐1, Jagged‐2 and Delta‐like‐1. We also provide evidence that contact with Notch ligands on thymic epithelium is necessary to activate and sustain Notch signaling in thymocytes, and that this can occur independently of positive selection induction. Our data suggest that Notch ligand expression by thymic epithelium may partly explain the specialization of these cells in supporting thymocyte development, by regulating Notch activation via an inductive signaling mechanism independently of signaling leading to positive selection.

Overexpression of ICAT highlights a role for catenin-mediated canonical Wnt signalling in early T cell development

Transcription factors of the T cell factor/lymphoid enhancing factor (Tcf/Lef) family are key regulators in the development of T cell precursors to the CD4+8+ stage. These factors are known targets of the canonical Wnt signalling pathway, and regulate transcription of Wnt target genes following interaction with the armadillo repeat‐containing protein β‐catenin. However, as recent studies show normal thymocyte maturation in the absence of either β‐catenin or its homologue γ‐catenin, the role of Wnt signalling in Tcf/Lef activation during T cell development is controversial. To directly investigate the importance of catenin‐mediated Wnt signalling in early thymocytes, we have compared the expression of β‐ and γ‐catenin and analysed distinct stages of T cell precursor maturation following overexpression of inhibitor of β‐catenin and Tcf (ICAT), which inhibits Wnt signalling by preventing binding of armadillo repeat‐containing proteins to Tcf/Lef. By direct retroviral gene targeting of CD4–8– and CD4+8+ precursors, we show that ICAT overexpression inhibits the CD4–8–‐to‐CD4+8+ transition, but not the CD4+8+‐to‐CD4+8– or ‐CD4–8+ transition. Collectively, our data support a model in which canonical Wnt signalling influences T cell development in the thymus by playing an essential role in the maturation of CD4–8– but not CD4+8+ thymocytes.

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.

Wnt-4 protects thymic epithelial cells against dexamethasone-induced senescence

Glucocorticoids are widely used immunosuppressive drugs in treatment of autoimmune diseases and hematological malignancies. Glucocorticoids are particularly effective immune suppressants, because they induce rapid peripheral T cell and thymocyte apoptosis resulting in impaired T cell-dependent immune responses. Although glucocorticoids can induce apoptotic cell death directly in developing thymocytes, how exogenous glucocorticoids affect the thymic epithelial network that provides the microenvironment for T cell development is still largely unknown. In the present work, we show that primary thymic epithelial cells (TECs) express glucocorticoid receptors and that high-dosage dexamethasone induces degeneration of the thymic epithelium within 24 h of treatment. Changes in organ morphology are accompanied by a decrease in the TEC transcription factor FoxN1 and its regulator Wnt-4 parallel with upregulation of lamina-associated polypeptide 2α and peroxisome proliferator activator receptor γ, two characteristic molecular markers for adipose thymic involution. Overexpression of Wnt-4, however, can prevent upregulation of adipose differentiation-related aging markers, suggesting an important role of Wnt-4 in thymic senescence.

Retinoid receptor-activating ligands are produced within the mouse thymus during postnatal development

Vitamin A deficiency is known to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Experimental evidence suggests that the active metabolites of vitamin A that mediate its effects on the immune system are the retinoic acids (RA), which are ligands for the nuclear RA receptor (RAR) family. RA were previously shown both to promote proliferation and to regulate apoptosis of thymocytes. In this study we detected the age‐dependent mRNA expression of retinaldehyde dehydrogenases (RALDH1 and 2), cellular RA binding protein‐II and CYP26A, proteins responsible for the synthesis, nuclear transport and degradation of RA in the postnatally developing thymus. RALDH1 was located in thymic epithelial cells. However, the amount of all‐trans RA in thymic homogenates was close to the detection limit, suggesting that in this tissue all‐trans RA is not the main RAR‐regulating product of retinol metabolism. At the same time, by measuring the induction of a RAR‐responsive transgene in two independent transgenic mouse strains, we demonstrated the production of an RAR‐activating ligand, which was age and RALDH dependent. Our data provide evidence for the existence of endogenous retinoid synthesis in the thymus and suggest that retinoids similar to glucocorticoids might indeed be involved in the regulation of thymic proliferation and selection processes by being present in the thymus in functionally effective amounts.

Con A activates an Akt/PKB dependent survival mechanism to modulate TCR induced cell death in double positive thymocytes

While low avidity ligation of the T cell receptor (TCR) leads to positive selection and further maturation of developing thymocytes providing the immune system with mature CD4(+) and CD8(+) (single positive) T cells, high avidity ligation triggers negative selection by apoptotic cell death and therefore the TCR repertoire is purged of autoreactive T cells. On peripheral T cells, however, high avidity ligation of the TCR triggers activation and survival not death. In the present study we used concanavalin A (Con A) and alpha-CD3 epsilon antibody to investigate a possible survival mechanism in connection with TCR ligation. Con A and alpha-CD3 epsilon were used in the study for the following reasons: (1) they both mimic the effects of high avidity TCR ligation by activating peripheral T cells, and (2) they trigger distinctively different physiological changes in developing thymocytes. While Con A supports events associated with cellular survival, alpha-CD3 epsilon induces apoptotic cell death. In our experimental system the TCR was cross-linked by Con A and alpha-CD3 epsilon in thymocytes of major histocompatibility complex (MHC) deficient thymus organ cultures, where signals from the TCR can be triggered on zero background signal level. We have found that TCR cross-linking by Con A and not by alpha-CD3 epsilon decreases the gene and protein expression of the pro-apoptotic molecule, Bad; and that Con A is capable of the activation of the survival signalling pathway including protein kinase B (Akt/PKB) independently of phosphatidyl inositol kinase (PI3K).

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.