No description available.

No description available.

Tracheobronchial submucosal glands (SMGs) develop from clusters of epithelial progenitor cells basally orientated within the surface airway epithelium called primordial glandular placodes (PGPs). Signal transduction events that coordinate the transitional process from PGPs into fully developed SMGs consisting of intricately branched networks of tubular secretary structures are still poorly understood. Wnt/β-catenin dependent induction of lymphoid enhancing factor-1 (Lef-1) expression in PGP progenitor/stem cells is required for SMG formation and maturation in the airway. In an effort to better understand the regulatory mechanisms that control Lef-1 during airway SMG development, I have studied its transcriptional regulation. I discovered that Sox2 expression is predominantly confined to the surface airway epithelium (SAE) and is repressed as Lef-1 is induced within PGPs. Deletion of Sox2 in polarized primary airway epithelia significantly enhances Lef-1 mRNA expression. Consequently, my hypothesis is that Sox2 functions as a negative regulator of Lef-1 expression in the SAE. I demonstrated that Sox2 modulates the expression of Lef-1 both independent and dependent on Wnt/β-catenin signaling. I discovered that a Sox2-binding site located in the Wnt Responsive Element (WRE) region of the 2.5Kb Lef-1 promoter is required for Sox2-mediated inhibition of β-catenin-dependent Lef-1 promoter transcription. It is important to understand the biology of SMG development because SMGs are the major mucus-producing structures in the proximal airway and are important in regulating the innate immunity of the lung in response to various neural signals. SMG ducts have also been proposed as a potential protective niche for slowly cycling progenitor cells (SCPCs). Hence, aberrant SMG function is thought to aggravate the pathoprogression of lung disease. Cystic fibrosis (CF) is a disease caused by a defect in the gene that encodes a chloride ion channel called cystic fibrosis transmembrane conductance regulator (CFTR). The absence of CFTR in serous cells within SMG ducts contributes to defective airway secretion, which alters the microenvironment within SMGs. I hypothesized that the glandular SCPC niche may be dysfunctional in CF. I reported that the neural peptide, calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. CFTR-deficient mice failed to maintain glandular SCPCs following airway injury, suggesting that the glandular SCPC niche may be dysfunctional in CF. CGRP levels increase following airway injury and function as an injury-inducible mitogen that stimulates progenitor cell proliferation. However, components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation through paracrine mechanisms. This discovery may have important implications for injury/repair mechanisms in the CF airway.

CD4+ and CD8+ T cells, the essential mediators of cellular immune responses, are produced in the thymus following sequential maturation stages. Hematopoietic progenitors first seed the thymus and make T cell lineage specification and commitment decisions within the CD4−CD8− double negative (DN) compartment. Thymocytes then mature to the CD4+CD8+ double positive (DP) stage, followed by vigorous negative and positive selection processes. The positively selected DP thymocytes first give rise to CD4+CD8lo intermediate (IM) cells which then differentiate into MHC class II-restricted CD4+ and MHC class I-restricted CD8+ T cells, a crucial decision known as CD4+ vs. CD8+ lineage choice. The lineage choice decision is influenced by the timing, intensity, and duration of signals derived from the TCR and cytokines, and recent studies have identified a number of transcriptional factors that intrinsically regulate this critical fate decision. Among these, Th-POK (encoded by Zbtb7b, called Thpok here for simplicity and consistency with the literature) is specifically required for CD4+ differentiation while Runx factors promote CD8+ T cell production and repress Cd4 in CD8+ lineage committed cells. Upregulation of Thpok is most evident in the CD4+8lo IM cells and is required to antagonize Runx3 activity and expression to promote CD4+ lineage commitment. Collectively, the Th-POK-Runx3 axis appears to be a critical convergence point in the CD4+ vs. CD8+ lineage choice. After committing to either CD4+ or CD8+ thymocytes, lineage-inappropriate genes are silenced to ensure the distinct identity and functional divergence between these two cell types. Repression of the Cd4 gene on CD8+ lineage committed cells is mediated by a ~430 bp silencer sequence in its first intron. Likewise, Thpok is repressed in CD8+ T cells by a ~560 bp sequence upstream of the Thpok exon 1a, and both Cd4 and Thpok silencers contain consensus binding motifs for Runx factors, which are necessary for CD8+ lineage commitment. T cell factor 1 (TCF-1) and lymphoid enhancer binding factor 1 (LEF-1) are members of the TCF-LEF family transcription factors and abundantly expressed in T lineage cells, and known to be necessary for the maturation of DN T cells to the DP stage. However, because germline deletion of TCF-1 and LEF-1 causes severe early T cell developmental block and embryonic lethality, respectively, their roles beyond the DP stage are unknown. In my thesis work, I overcame these obstacles by conditionally ablating both TCF-1 and LEF-1 in DP thymocytes using CD4-Cre. We observed impaired differentiation of CD4+ T cells from the bipotent DP precursors in the absence of TCF-1 and LEF-1. Mechanistically, TCF-1 promotes CD4+ T cell development by positively regulating the expression of Thpok. TCF-1 and LEF-1 deficiency also results in derepression of the CD4 co-receptor in CD8+ lineage committed cells. In CD8+ T cells, TCF-1 interacts with Runx3 to repress expression of Cd4. These findings not only broaden the spectra of TCF-LEF-mediated regulatory activities in late stages of T cell development, but also reveal new paradigms in T cell fate decision and identity maintenance.

La transició epiteli-mesènquima (TEM) és un procés en què cèl lules epitelials, immòbils i amb polaritat apico-basal transiten cap un fenotip mesenquimal o fibroblàstic. L'expressió del factor de transcripció snail1 és suficient per induir TEM en cèl lules en cultiu i és necessari per la majoria de les TEM fisiològiques descrites. Snail1 és un membre de la família de proteïnes amb dits de Zinc que reprimeix gens epitelials (com l'E-cadherina) a través de la unió directa a seqüències especifiques dels promotors anomenades caixes E i posterior reclutament de corepressors. La TEM també es caracteritza per l'activació de gens mesenquimals, però el mecanisme pel qual snail1 indueix l'expressió d'aquests és poc conegut. En aquest treball demostrem que snail1 actua a nivell transcripcional per incrementar els nivells dels marcadors mesenquimals FN1 (fibronectina) i LEF1 (de l'anglès, lymphoid enhancer-binding factor 1) a través d'un mecanisme nou per aquesta proteïna de dits de Zn que no requereix ni caixes E ni unió directa a l'ADN. A més a més, mostrem que, per a dur a terme l'activació, snail1 coopera amb dos factors de transcripció ja descrits en relació a la TEM: beta-catenina i NF-kappa-B. Els nostres resultats també proven que l'expressió forçada de la E-cadherina evita aquesta cooperació i conseqüent activació gènica. A banda d'aquest mecanisme, també hem descrit que el factor de transcripció TFCP2c, que no havia estat prèviament relacionat amb TEM, és necessari per l'activació del gen FN1 induïda per snail1. / Epithelial-mesenchymal transition (EMT) is a cellular process by which no motile epithelial, apico-basal-polarized cells transit towards a motile mesenchymal front-backpolarized phenotype. Expression of the transcription factor snail1 is sufficient to induce EMT in cultured cells and it is required for most of the physiological EMTs described. Snail1 is a member of the Zn finger protein family that represses epithelial genes (such as E-cadherin) by directly binding to specific promoter sequences called E-boxes and subsequent recruitment of corepressors. EMT is also accompanied by activation of mesenchymal genes, however, little is known of how snail1 induces their expression.In this work we provide evidence that snail1 acts at the transcriptional level to increase the levels of the mesenchymal FN1 (fibronectin) and LEF1 (lymphoid enhancer-binding factor 1) genes through a novel mechanism for this Zn finger protein that does not require neither E-boxes nor direct binding to DNA. Furthermore, we describe a cooperative action in such mechanism between snail1 and two transcription factors previously related to EMT: beta-catenin and NF-kappaB. Our results also show that restoration of E-cadherin levels prevents such cooperation and subsequent activation. In addition, we also demonstrate that TFCP2c, which had not been previously linked to EMT, is also required for snail1-induced transcriptional activation of the FN1 gene.