Biology subjects

Cardoso, W. V.

Publications and source records attributed to Cardoso, W. V..

8 recordsLinked to original sources

bioRxiv2025

Genetic background and transient prenatal disruption of vitamin A signaling determine susceptibility to airway hyperresponsiveness in mice

Airway structural changes and hyperresponsiveness (AHR), hallmarks of asthma, are crucially influenced by genetic variations and adverse exposures. While intrauterine environmental perturbations leading to dysfunctional lung development have been linked to adult pulmonary disease, still little is known about the developmental events leading to these postnatal abnormalities. Here, we provide evidence of genetic background playing a key role in this process. Using A/J and C57BL/6J mice known for their distinct susceptibility to AHR, we show that A/J but not C57BL/6J develop an aberrant airway smooth muscle (SM) program and AHR in adulthood when exposed transiently to a vitamin A/retinoic acid (RA)-disrupted intrauterine environment in vivo by a maternal BMS493 administration. Single nuclei multiomics analysis identified a subpopulation of mesenchymal cells that overactivated TGF{beta} targets in response to BMS selectively in A/J, but not C57BL/6J, embryonic lungs. These cells, localized to sites of airway SM initiation, exhibited robust BMS-mediated upregulation of SMAD2/3 targets, including regulators of SM program Pdgfra and Tnc, and showed stable cell proportions despite the marked transcriptional rewiring following RA disruption. These findings identify TGF{beta}-activating mesenchymal cells as a critical niche responsive to RA signaling and reveal how genetic background determines developmental susceptibility to micronutrient perturbations with long-term impact on airway function.

developmental biology↗

bioRxiv2025

Allosteric inhibition rescues hydrocephalus caused by catalytically inactive Shp2

SHP2, a protein tyrosine phosphatase (PTP) crucial in Ras-MAPK signaling, is associated with various human congenital diseases and cancers. Here, we show that the catalytically inactive Shp2C459S mutation results in communicating hydrocephalus, similar to the catalytically activating Shp2E76K and Mek1DD mutants. Unlike previous mutants, however, Shp2C459S/+ mutation uniquely affects ciliary development rather than neurogenesis, leading to reduced cilia density and impaired ciliary motility. Differential scanning fluorimetry revealed that SHP2C459S, SHP2E76K and SHP2C459S/E76K mutations all induce an open SHP2 conformation, but only SHP2C459S leads to aberrant GAB1 phosphorylation in cells expressing wild-type SHP2. This distinctive signaling pattern correlates with our observations in brain ventricular tissues of Shp2C459S/+ mice, where Erk and Stat3 activities remain normal but Gab1 phosphorylation is elevated. Critically, we show that the hydrocephalus phenotype in Shp2C459S mice can be mitigated by allosteric inhibition of Shp2. These findings suggest that Shp2-associated hydrocephalus is driven by conformational changes rather than altered catalytic activity. Our results underscore the therapeutic potential of conformation-specific allosteric inhibitors in targeting both catalytically active and inactive SHP2 mutants.

pathology↗

bioRxiv2024

Identification and Targeting of Regulators of SARS-CoV-2-Host Interactions in the Airway Epithelium.

Although the impact of SARS-CoV-2 in the lung has been extensively studied, the molecular regulators and targets of the host-cell programs hijacked by the virus in distinct human airway epithelial cell populations remain poorly understood. This is in part ascribed to the use of nonprimary cell systems, overreliance on single-cell gene expression profiling that does not ultimately reflect protein activity, and bias toward the downstream effects rather than their mechanistic determinants. Here we address these issues by network-based analysis of single cell transcriptomic profiles of pathophysiologically relevant human adult basal, ciliated and secretory cells to identify master regulator (MR) protein modules controlling their SARS-CoV-2-mediated reprogramming. This uncovered chromatin remodeling, endosomal sorting, ubiquitin pathways, as well as proviral factors identified by CRISPR analyses as components of the host response collectively or selectively activated in these cells. Large-scale perturbation assays, using a clinically relevant drug library, identified 11 drugs able to invert the entire MR signature activated by SARS-CoV-2 in these cell types. Leveraging MR analysis and perturbational profiles of human primary cells represents a novel mechanism-based approach and resource that can be directly generalized to interrogate signatures of other airway conditions for drug prioritization.

cell biology↗

bioRxiv2024

Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

developmental biology↗

bioRxiv2022

Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation

Basal cells are multipotent stem cells of a variety of organs, and in the lung are known as crucial components of the airway epithelium. However, it remains unclear how diverse basal cells are and whether distinct subpopulations respond differently to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the murine trachea. Among these we found two major subpopulations comprising the most stem-like progenitor compartment, but with distinct signatures and ability to self-renew and differentiate. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to initiate an aberrant program of differentiation in response to environmental perturbations in primary cultures and in injury mouse models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of cartilage-deficient mouse mutants. Lastly, we show that key features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide critical insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

developmental biology↗

bioRxiv2022

Alveolar regeneration following viral infection is independent of tuft cells

Severe injuries following viral infection cause lung epithelial destruction with the presence of ectopic basal progenitor cells (EBCs), although the exact function of EBCs remains controversial. We and others previously showed the presence of ectopic tuft cells in the disrupted alveolar region following severe influenza infection. Here, we further revealed that the ectopic tuft cells are derived from EBCs. This process is amplified by Wnt signaling inhibition but suppressed by Notch inhibition. Further analysis revealed that p63-CreER labeled population de novo arising during regeneration includes alveolar epithelial cells when Tamoxifen was administrated after viral infection. The generation of the p63-CreER labeled alveolar cells is independent of tuft cells, demonstrating segregated differentiation paths of EBCs in lung repair. EBCs and ectopic tuft cells can also be found in the lung parenchyma post SARS-CoV-2 infection, suggesting a similar response to severe injuries in humans.

cell biology↗

bioRxiv2022

A local translation program regulates centriole amplification in the airway epithelium

Biogenesis of organelles requires targeting of a subset of proteins to specific subcellular domains by signal peptides or mechanisms controlling mRNA localization and local translation. How local distribution and translation of specific mRNAs for organelle biogenesis is achieved remains elusive and likely to be dependent on the cellular context. Here we identify Trinucleotide repeat containing-6a (Tnrc6a), a component of the miRNA pathway, distinctively localized to apical granules of differentiating airway multiciliated cells (MCCs) adjacent to centrioles. In spite of being enriched in TNRC6A and the miRNA-binding protein AGO2, they lack enzymes for mRNA degradation. Instead, we found these apical granules enriched in components of the mRNA translation machinery and newly synthesized proteins suggesting that they are specific hubs for target mRNA localization and local translation in MCCs. Consistent with this, Tnrc6a loss of function prevented formation of these granules and led to a broad reduction, rather than stabilization of miRNA targets. These included downregulation of key genes involved in ciliogenesis and was associated with defective multicilia formation both in vivo and in primary airway epithelial cultures. Similar analysis of Tnrc6a disruption in yolk sac showed stabilization of miRNA targets, highlighting the potential diversity of these mechanisms across organs. HighlightsO_LITnrc6a is expressed in the lung selectively in differentiating multiciliated cells (MCC) adjacent to centrioles. C_LIO_LITNRC6A localizes to apical granules containing AGO2, miRNAs and their targets, but lacking mRNA degradation enzymes. C_LIO_LITNRC6A granules are enriched in components of the mRNA translation machinery and show evidence of concentrated newly-synthesized proteins C_LIO_LILoss of Tnrc6a in the lung leads to reduction, not stabilization of miRNA targets. C_LIO_LITnrc6a is required for efficient centriole amplification and multicilia formation. C_LI

cell biology↗

bioRxiv2022

Prom1 and Notch regulate ciliary length and dynamics in multiciliated cells of the airway epithelium

Differences in ciliary morphology and dynamics among multiciliated cells of the respiratory tract have been well reported and known to contribute to efficient mucociliary clearance. Nevertheless, little is known about how phenotypic differences among multiciliated cells are established in the mammalian lung. Here we show that Prominin-1 (Prom1), a transmembrane protein widely used as stem cell and tumor-initiating marker, is crucial to this process. During airway differentiation, Prom1 becomes restricted to multiciliated cells, where it is expressed at distinct levels along the proximal-distal axis of the airways and in the adult airway epithelium in vitro. We found that Prom1 is induced by Notch in post-specified multiciliated cells and that Notch inactivation abolishes the gradients of Prom1 in the developing airways and in differentiating organotypic cultures. Prom1 was not required for multicilia formation and when inactivated resulted in longer cilia, which remained functional but beating at a lower frequency. Disruption of Notch resulted in opposite effects and suggested that Notch fine-tunes Prom1 levels to regulate the multiciliated cell phenotype and generate diversity among these cells in the respiratory tract. By controlling these features, this mechanism contributes to the innate defense of the lung against environmental agents and prevent pulmonary disease. Significance StatementMulticiliated cells are integral components of the epithelia from a variety of organs. In the respiratory tract they are crucial for mucociliary clearance, a first line of defense against environmental agents and microorganisms. Regional differences in ciliary morphology and dynamics of multiciliated cells have been well described. However, little is known about the events generating phenotypical and functional differences among these cells in airways. Here we provide evidence of a novel mechanism in post-specified multiciliated progenitors whereby local Notch and Prom1 regulate ciliary length and ciliary beating to generate morphological and functional diversity among the multiciliated cells. The findings provide insights into the impact of these signals in maintaining the integrity and function of the airway epithelium, preventing pulmonary disease.

cell biology↗