Gli family zinc finger 1 is associated with endothelin receptor type B in Hirschsprung disease

Abstract

Hirschsprung disease (HSCR) is a newborn colorectal disease characterized by an absence of ganglia in the distal gut. Hedgehog (Hh) and endothelin signaling serve important roles in gastrointestinal tract formation. Alterations in the signaling pathways disrupt the development of enteric neural crest cells (ENCCs). It is not known whether there is any coordination between these pathways in the pathogenesis of HSCR. In the present study, tissue samples from 35 patients with HSCR, including stenotic aganglionosis gut and normal ganglionic gut, were obtained. The expression of Gli family zinc finger 1 (Gli1) and endothelin receptor type B (EDNRB) was determined using reverse transcription‑quantitative polymerase chain reaction, immunohistochemistry and western blotting. In addition, the SK‑N‑SH cell line was used to investigate the association between Hh signaling and the expression of EDNRB. The results revealed aberrant expression of Gli1 in the aganglionic segments, as well as decreased expression of Gli1 in tissues from 7 patients with HSCR exhibited, whereas tissues from 9 patients with HSCR exhibited increased Gli1 expression compared with the expression in the normal tissues. There was a negative association between EDNRB expression and Gli1 expression in the same sample. Knockdown of Gli1 by small interfering RNA and inhibition of Hh signaling by Vismodegib in SK‑N‑SH cells increased EDNRB expression. By contrast, upregulation of Gli1 expression by plasmids and activation of Hh signaling by Purmorphamine decreased EDNRB expression. Furthermore, premature enteric ganglia were observed in 4 patients with HSCR with decreased Gli1 expression. Thus, the results of the present study suggest that altered Gli1 expression negatively regulates EDNRB expression in patients with HSCR. The increased expression of EDNRB induced by decreased Gli1 expression may represent a novel mechanism in HSCR.

Keywords: Hirschsprung disease; hedgehog signaling pathway; endothelin receptor type B; enteric neural crest cells.

Figure 1.

Gli1 and EDNRB expression in tissues from patients with HSCR. Upper and lower panels indicating Gli1 and EDNRB expression in (A) a low-level Gli1 expression sample from patient #32 and (B) a high-level Gli1 expression sample from patient #27 using immunohistochemistry. Magnification, ×100; brown color, targeted proteins; blue color, nucleus; Representative western blot analysis of Gli1 and EDNRB expression in (C) low-level Gli1 samples from patient #32 and #35 and (D) high-level Gli1 specimens from patient #20 and #27, respectively. (E) Scatter plots for corresponding expression of Gli1 and EDNRB mRNA in (E) low-level Gli1 samples (n=7) and (F) high-level Gli1 samples (n=9), respectively. Values are expressed as the mean ± standard deviation. **P<0.01. Gli1, Gli family zinc finger 1; EDNRB, endothelin receptor type B; HSCR, Hirschsprung disease.

Figure 2.

Hh negatively regulates EDNRB expression in the SK-N-SH cell line. (A) Protein expression of Gli1 and EDNRB in SK-N-SH cells following transfection with Gli1 siRNA and negative control siRNA using western blot analysis. (B) Protein expression of Gli1 and EDNRB in SK-N-SH cells stably transfected with Gli1 plasmid and control vector. (C) RT-qPCR and (D) western blot analysis were performed on cells treated with Vismodegib (5, 10 and 20 µM) or DMSO for 48 h. (E) RT-qPCR and (F) western blot analysis were performed on cells treated with Purmorphamine (2.5, 5 and 10 µM) or DMSO for 48 h. Values are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. DMSO groups. Hh, hedgehog; EDNRB, endothelin receptor type B; Gli1, Gli family zinc finger 1; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; DMSO, dimethyl sulfoxide.

Figure 3.

H&E staining indicating ganglia in tissues from patients with HSCR. Upper panels indicate H&E results for ganglia in a low-level Gli1 expression sample from patient #32. Left image indicates mature ganglia in normal gut tissues and right image indicates premature ganglia in stenotic gut tissues. Lower panels indicate H&E results for ganglia in a high-level Gli1 expression specimen from patient #20. Left image indicates mature ganglia in normal gut tissue and right image indicates a lack of ganglia in stenotic gut tissue. Magnification, ×200. H&E, hematoxylin and eosin; HSCR, Hirschsprung disease; Gli1, Gli family zinc finger 1.