Identification of multiple isoforms of glucocorticoid receptor in nasal polyps of patients with chronic rhinosinusitis

Abstract

Background: The conventional belief that glucocorticosteroid (GC) acts through a single brand glucocorticoid receptor (GR)α protein has changed dramatically with the discovery of multiple GR isoforms. We aimed to evaluate whether multiple GR protein isoforms are expressed in chronic rhinosinusitis with nasal polyps (CRSwNP) and whether GR protein isoform expression profiles differ between different endotypes of CRSwNP.

Methods: Thirty-eight patients with CRSwNP and ten healthy volunteers were included. The protein expression of multiple GR isoforms in nasal polyps (NPs) tissue and control mucosae was examined by western blot analysis with different GR antibodies.

Results: Five bands, including three bands for known proteins (GRα-A/B, GRα-C, and GRα-D) and two bands for unidentified proteins at 67 kilodaltons (kDa) and 60 kDa, were identified with both total GR antibody (PA1-511A) and GRα-specific antibody (PA1-516). GRα-D intensity, which was abundant in nasal mucosa, was significantly increased in the CRSwNP group and was especially elevated in the noneosinophilic CRSwNP (NE-CRSwNP) group (PA1-511A: P < 0.001 and P = 0.0018; PA1-516: P < 0.003 and P = 0.006, respectively). Additionally, the intensities of the newly recognized 67 kDa and 60 kDa bands were much greater in the NE-CRSwNP subgroup than in the eosinophilic CRSwNP (E-CRSwNP) subgroup; in the E-CRSwNP subgroup, the median intensities were even lower than those in the control group.

Conclusions: This study provides evidence that nasal tissues express multiple GR protein isoforms. GR protein isoforms presented disease and tissue-specific expression profiles that differed between the CRSwNP and control groups and between the E-CRSwNP and NE-CRSwNP subgroups.

Keywords: Endotypes; Glucocorticoid receptor; Nasal polyps; Sinusitis.

Fig. 1

Western blots of representative GR bands in the CRSwNP group and control group. NP tissues and control mucosae were probed with a an anti-total GR antibody (PA1-511A) and b an anti-GRα antibody (PA1-516). The images showed five GR bands of approximately 91–94, 80, 67, 60 and 52 kDa. β-Actin was used as an internal control. GR glucocorticoid receptor, CRSwNP chronic rhinosinusitis with nasal polyps, kDa kilodaltons

Fig. 2

Expression profiles of representative GR isoforms between the CRSwNP group and the control group. The relative expression levels of a the sum of bands 1–5 and representative bands for GRs, including b GRα-A/B, c GRα-C, d the 60 kDa protein and e GRα-D, which were detected with an anti-total GR antibody (PA1-511A), were analyzed in the CRSwNP group compared to the control group. Similar band intensity comparisons were performed to analyze f the sum of bands 1–5 and the bands for g GRα-A/B, (H) the 67 kDa protein, and i GRα-D, which were detected with an anti-GRα antibody (PA1-516). Data was presented as a ratio of each isoform to an internal control which were relative to β-actin expression. The data were presented in dot plots with medians and interquartile ranges. Nonparametric tests (Mann–Whitney tests) were used for comparisons between these two groups. GR, glucocorticoid receptor; CRSwNP, chronic rhinosinusitis with nasal polyps; kDa, kilodaltons

Fig. 3

Expression profiles of representative GR isoforms among subgroups representing different endotypes of CRSwNP and the control group. The relative expression levels of a the sum of bands 1–5 and representative bands for GRs, including b GRα-A/B, c GRα-C, d the 60 kDa protein and e GRα-D, which were detected with an anti-total GR antibody (PA1-511A), were analyzed among the E-CRSwNP subgroup, the NE-CRSwNP subgroup and the control group. Similar band intensity comparisons were performed to analyze f the sum of bands 1–5 and the bands for g GRα-A/B, (H) the 67 kDa protein, and i GRα-D, which were detected with an anti-GRα antibody (PA1-516). Data was presented as a ratio of each isoform to an internal control which were relative to β-actin expression. The data were presented in dot plots with medians and interquartile ranges. GR, glucocorticoid receptor; CRSwNP, chronic rhinosinusitis with nasal polyps; kDa, kilodaltons; E-NP, eosinophilic nasal polyp; NE-NP, noneosinophilic nasal polyp

Fig. 4

Percent expression of GR isoforms in subgroups representing different endotypes of CRSwNP and the control group. The isoforms were detected with a an anti-total GR antibody (PA1-511A) and b an anti-GRα antibody (PA1-516) in the subgroups representing different endotypes of CRSwNP and the control group. The percentages were calculated as the relative expression levels of individual GR isoforms compared to the sum of expression levels for bands 1–5. The pie charts displayed the medians for all individuals in either the different endotype subgroups of the CRSwNP group or the control group. GR, glucocorticoid receptors; CRSwNP, chronic rhinosinusitis with nasal polyps; kDa, kilodaltons; E-NP, eosinophilic nasal polyp; NE-NP, noneosinophilic nasal polyp