Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease - PubMed (original) (raw)

Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease

Ann E Tilley et al. Am J Respir Crit Care Med. 2009.

Abstract

Rationale: The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults.

Objectives: We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD).

Methods: We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR.

Measurements and main results: Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers.

Conclusions: These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

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Figures

**Figure 1.

The Notch pathway in mammals. The pathway is triggered by the Delta-like or Jagged ligands interacting with one or more of the Notch receptors. The pathway is activated by intracellular cleavage of a Notch receptor, releasing the Notch intracellular domain, which translocates to the nucleus where it activates CBF1 (gene symbol, RBPSUH), the primary transcription factor to activate downstream Notch effector pathways (Hes1, 2, 5; Hey1, 2 and L). A variety of transcriptional coactivators and cytoplasmic and nuclear modulators tune the pathway, providing multiple points of control. When Notch signaling is active, differentiation is suppressed. When Notch signaling is turned “off,” differentiation to specific cell fates is allowed to proceed.

**Figure 2.

Relative expression of Notch ligands, receptors, and downstream effectors in the small airway epithelium of normal nonsmokers (n = 12). TaqMan quantitative real-time reverse transcriptase polymerase chain reaction was used to generate relative expression data for Notch-related ligands, receptors, and downstream effector genes. Each bar represents the mean expression level ± standard error for the gene in nonsmokers small airway epithelium.

**Figure 3.

Immunostaining for key Notch proteins in airway epithelial biopsies. (A) Chronic obstructive pulmonary disease (COPD) smoker, goat anti-human Jag1 antibody. (B) COPD smoker, goat anti-human Jag1 antibody plus Jag1 peptide. (C) COPD smoker, rabbit anti-human Notch2 antibody. (D) COPD smoker, rabbit anti-human Notch2 antibody plus Notch2 peptide. (E) Normal nonsmoker, rabbit anti-human Hes1 antibody. (F) Normal nonsmoker, rabbit anti-human Hes1 antibody plus Hes1 peptide. (G) Normal smoker, rabbit anti-human Hes5 antibody. (H) Normal smoker, rabbit IgG control.

**Figure 4.

Immunohistochemistry assessment of Hes1 protein expression in brushed small airway epithelial cells. (A) Ciliated cell, nonsmoker, rabbit anti-Hes1 antibody. (B) Ciliated cell, nonsmoker, rabbit anti-Hes1 antibody. Note the variability in Hes1 abundance in 2 cells from the same individual. (C) Undifferentiated cell (left) and secretory cell (right), nonsmoker, rabbit anti-Hes1 antibody. (D) Secretory cell, nonsmoker, rabbit anti-Hes1 antibody. (E) Secretory cell, nonsmoker, rabbit anti-Hes1 antibody. (F) Basal cells, nonsmoker, rabbit anti-Hes1 antibody. (G) Basal cell, nonsmoker, rabbit anti-Hes1 antibody. (H) Ciliated cell, nonsmoker, rabbit anti-Hes1 antibody plus Hes1 peptide control. (I) Ciliated cell, nonsmoker, rabbit anti-Hes1 antibody plus Hes1 peptide control. Bar, 20 μm.

**Figure 5.

Immunohistochemistry assessment of Hes5 protein expression in brushed small airway epithelial cells. (A) Ciliated cell, nonsmoker, rabbit anti-Hes5 antibody. (B) Ciliated cell, nonsmoker, rabbit anti-Hes5 antibody. (C) Secretory cell, COPD smoker, rabbit anti-Hes5 antibody. (D) Secretory cell, COPD smoker, rabbit anti-Hes5 antibody. (E) Basal cell, nonsmoker, rabbit anti-Hes5 antibody. (F) Basal cells, nonsmoker, rabbit anti-Hes5 antibody. (G) Undifferentiated cell, COPD smoker, rabbit anti-Hes5 antibody. (H) Undifferentiated cell, nonsmoker, rabbit anti-Hes5 antibody. (I) Ciliated cells, nonsmoker rabbit IgG control. (J) Ciliated cells, nonsmoker rabbit IgG control. Bar, 20 μm.

**Figure 6.

Comparison of the relative expression of selected ligands of the Notch pathway in nonsmokers (n = 12), normal smokers (n = 15), and smokers with chronic obstructive pulmonary disease (COPD) (n = 13). TaqMan polymerase chain reaction was used to generate the data. Each bar represents mean expression with standard error; P values are represented in brackets above the bars.

**Figure 7.

Comparison of the relative expression of selected receptors of the Notch pathway in nonsmokers (n = 12), normal smokers (n = 15), and smokers with COPD (n = 13). TaqMan PCR was used to generate the data. Each bar represents mean expression with standard error; P values are represented in brackets above the bars.

**Figure 8.

Comparison of the relative expression of selected downstream effectors of the Notch pathway in nonsmokers (n = 12), normal smokers (n = 15), and smokers with chronic obstructive pulmonary disease (COPD) (n = 13). TaqMan polymerase chain reaction was used to generate the data. Each _bar_represents mean expression with standard error; P values are represented in brackets above the bars. Note that the scale is different for Hes1 compared with Hes5, Hey1, and Hey2, reflecting the higher relative level of expression of Hes1 compared with the other downstream effectors.

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Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease - PubMed (original) (raw)