14 research outputs found
Isolation and Characterization of the Human CP49 Gene Promoter
PURPOSE. This study focuses on the identification of regulatory elements that contribute to lens-specific expression of the human CP49 gene within the 5Š-flanking DNA sequences. METHODS. The DNA sequence upstream of the human CP49 coding region was subcloned as a set of 5Š and 3Š deletion series. The constructs were transfected into lens (N/N1003A) and nonlens (NIH3T3) cell lines and chicken primary lens cultures, to test for promoter activity and specificity. To further test the specificity, a portion of the 5Š flanking DNA sequence was used to drive transgene expression in mice. The flanking DNA sequence was analyzed for potential transcription factorbinding sites. RESULTS. The 5Š-flanking DNA preferentially activated reporter gene expression in a lens-preferred manner when transfected into cultured cells. Transgene expression driven by the CP49 promoter region was lens specific. Analysis of the proximal promoter sequence revealed the presence of potential binding sites for the AP-1, AP-2, and OCT-1 transcription factors and the absence of TATA and CAAT boxes. CONCLUSIONS. The sequence upstream of the CP49 gene possesses promoter activity and is able to drive lens-preferred expression in both transfection and transgenic experiments. Promoter activity is dependent on the presence of the proximal 300 bp directly upstream of the coding region. O cular lens development commences with a thickening of the anterior surface ectoderm that establishes the boundary of the presumptive lens as the lens placode. Invagination of the thickened ectoderm creates the lens pit, which is then pinched off from the ectoderm as a hollow sphere of cells known as the lens vesicle. The epithelial cells at the posterior end of the lens vesicle undergo terminal differentiation to form primary lens fiber cells. During this process, the cells elongate to fill the lumen of the lens vesicle. The anterior portion of the lens is lined by a single layer of epithelial cells that remain mitotically active and undergo differentiation at the equator, or bow region, of the lens to continually add secondary fiber cells to the lens mass. In addition to cell elongation the differentiation process of lens epithelial cells to lens fiber cells involves the loss of the nucleus and other membrane-bound organelles and alterations in gene expression. 1 Lens fiber cell formation results in the upregulation of a limited collection of genes that includes members of the soluble crystallin gene family, major intrinsic protein (MIP), and the core components of the lens fiber-specific beaded filament CP49 and CP115. 2 The CP49 and CP115 genes have been classified as divergent members of the intermediate filament family on the basis of conserved primary DNA sequence and gene structure. 7 Mutations in the CP49 have been linked to cataract formation, suggesting a role for the beaded filament, which is critical to lens clarity. 10 -13 The mutation or absence of any one of these factors in mice, accomplished through targeted gene deletion, results in abnormal eye and/or lens development, ranging from defective lens fiber cell differentiation to the complete absence of the lens. In this study, the 5Š-flanking and intronic DNA sequences of the human CP49 gene have been analyzed for potential regulatory elements. The transfection of cell lines and primary lens cultures and the production of transgenic animals were performed to test the proximal promoter for contributions to the cell type and differentiation-stage-specific expression pattern observed for the human CP49 gene. The results of these experiments suggest that the 5Š-flanking DNA proximal to the human coding sequence confers lens-preferred expression of reporter genes. Sequence analysis of the 5Š-flanking region does not identify consensus binding sites for transcription factors used in the regulation of crystallin genes, suggesting the possibility of separate control mechanism(s) for crystallin and noncrystallin gene regulation within the lens fiber cell
Differential Modulation of Keratin Expression by Sulforaphane Occurs via Nrf2-dependent and -independent Pathways in Skin Epithelia
In this study the authors address the mechanistic basis of the therapeutic benefit afforded by treatment with the naturally occurring small molecule sulforaphane in the context of a keratin-based skin blistering disease, epidermolysis bullosa simplex
Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population
Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis.
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Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population.
Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program
Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population.
Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program
Molecular mapping of interstitial lung disease reveals a phenotypically distinct senescent basal epithelial cell population
Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosisāassociated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program
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Osteopontin Links Myeloid Activation and Disease Progression in Systemic Sclerosis.
Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD
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Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis.
BackgroundThere is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF.MethodsGene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80).Results2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model).ConclusionsMicroscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF