61 research outputs found
Protocadherin-1 binds to SMAD3 and suppresses TGF-?1-induced gene transcription.
No full textGenetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-?1 (TGF-?1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o(-) (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-?1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-?1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-?-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-? target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-?-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-? signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-?
Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal
Get PDF17 p.-6 figRelatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand,replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly,autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.F.R. is supported by Marie Skłodowska-Curie Cofund [713660], Marie
Skłodowska-Curie ITN [765912], ALW Open Program [ALWOP.310]and ZonMW VICI [016.130.606] grants. R.C. and C.R. are supported by the Dutch Cancer Society [Grant number 5792 and 12458]. Patricia Boya is supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI),the Fondo Europeo de Desarrollo Regional (FEDER) [PGC2018-098557-B-I00] and a Marie Skłodowska-Curie ITN grant [765912]. Idil Orhon is a recipient of a FEBS postdoctoral fellowship and Beatriz Villarejo-Zori of a Fundacion Tatiana Perez de Guzman el Bueno predoctoral fellowhip.Peer reviewe
Protocadherin-1 localization and cell-adhesion function in airway epithelial cells in asthma
Get PDFBackgroundThe asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma.MethodsWe performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance.ResultsPCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding.ConclusionsIn conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair
Ambrisentan and Sildenafil mitigate early radiation-induced cardiopulmonary side-effects
No full textAmbrisentan and Sildenafil mitigate early radiation-induced cardiopulmonary side-effects
No full textAmbrisentan and Sildenafil mitigate early radiation-induced cardiopulmonary side-effects
No full textAmbrisentan and Sildenafil mitigate early radiation-induced cardiopulmonary side-effects
No full textThe impact of cardiac sparing on long-term pulmonary vascular recovery in proton-irradiated rats
No full textThe necessity of heart sparing for cardiopulmonary recovery after thoracic irradiation investigated in a rat model system of precise lung and heart proton irradiation
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