Transient activation of hedgehog pathway rescued irradiation-induced hyposalivation by preserving salivary stem/progenitor cells and parasympathetic innervation

Abstract

Purpose: To examine the effects and mechanisms of transient activation of the Hedgehog pathway on rescuing radiotherapy-induced hyposalivation in survivors of head and neck cancer.

Experimental design: Mouse salivary glands and cultured human salivary epithelial cells were irradiated by a single 15-Gy dose. The Hedgehog pathway was transiently activated in mouse salivary glands, by briefly overexpressing the Sonic hedgehog (Shh) transgene or administrating smoothened agonist, and in human salivary epithelial cells, by infecting with adenovirus encoding Gli1. The activity of Hedgehog signaling was examined by the expression of the Ptch1-lacZ reporter and endogenous Hedgehog target genes. The salivary flow rate was measured following pilocarpine stimulation. Salivary stem/progenitor cells (SSPC), parasympathetic innervation, and expression of related genes were examined by flow cytometry, salisphere assay, immunohistochemistry, quantitative reverse transcription PCR, Western blotting, and ELISA.

Results: Irradiation does not activate Hedgehog signaling in mouse salivary glands. Transient Shh overexpression activated the Hedgehog pathway in ductal epithelia and, after irradiation, rescued salivary function in male mice, which is related with preservation of functional SSPCs and parasympathetic innervation. The preservation of SSPCs was likely mediated by the rescue of signaling activities of the Bmi1 and Chrm1-HB-EGF pathways. The preservation of parasympathetic innervation was associated with the rescue of the expression of neurotrophic factors such as Bdnf and Nrtn. The expression of genes related with maintenance of SSPCs and parasympathetic innervation in female salivary glands and cultured human salivary epithelial cells was similarly affected by irradiation and transient Hedgehog activation.

Conclusions: These findings suggest that transient activation of the Hedgehog pathway has the potential to restore salivary gland function after irradiation-induced dysfunction.

Fig. 1

Effects of IR and Shh on Hh activity in SMGs. (A) SMG samples from Ptch1-lacZ Hh reporter transgenic mice were collected before (D0) or 3, 7 or 14 days after 15 Gy single dose IR, and analyzed with qRT-PCR for relative mRNA expression (n=3). (B) Krt5-rtTA/tetO-Shh mice were not treated (NT) or induced with Dox for 7 days, then sacrificed to collect SMGs for qRTPCR analysis of transgenes and genes related with Hh pathway (n=3). (C,D) SMGs of male Krt5-rtTA/tetO-Shh/Ptch1-lacZ mice were collected for X-gal staining on sections (C) or salispheres (D) without or with Dox induction for 7 days.

Fig. 2

Transient Hh activation rescued radiation-induced hyposalivation and expression of acinar markers. (A) The stimulated whole saliva flow rate in male Krt5-rtTA/tetO-Shh mice irradiated (IR) or IR and induced with Dox for 7 days starting from Day 0, 3 or 90 after IR (IR+Dox D0, D3 or D90) were standardized with the body weight and normalized to that of non-treated (NT) group (n = 5). (B-D) 120 days after IR, SMG samples were collected and analyzed with qRT-PCR (B, C) and Western blot (D) for expression of acini marker Aqp5 and Chrm3 normalized to Gapdh (n=3). *: P<0.05, **: P < 0.01.

Fig. 3

Transient Hh activation preserved functional SSPCs and the activity of Bmi1 pathway impaired by IR. (A) Flow cytometry analysis of c-Kit⁺/Sca-1⁺ cells in SMGs. (B) Salisphere numbers from each whole SMG collected 10 days after IR or 7 day after Dox treatment. (C) Expression of genes related with SSPCs and proliferation examined by qRT-PCR. (D) Flow cytometry analysis of Bmi1⁺ cells in SMGs . (E) Western blot and relative quantification of p21^Waf1. n=3 for all studies. ns: not significant.

Fig. 4

Transient Hh activation preserved parasympathetic innervation and the expression of neurotrophic factors impaired by IR. (A) AChE staining on SMG sections and the quantification of staining intensity. (B) Western blot and relative quantification of GFRα2. (C) Expression of neurotrophic factors and miR-206 examined by qRT-PCR. (D) Concentrations of Bdnf in whole saliva or SMG homogenates and that of SMG Nrtn detected by ELISA. n=3 for all studies.

Fig. 5

Transient Hh activation preserved the activity of Chrm1 pathway impaired by IR. (A) Expression of miR-107, Chrm1 and Ccna2 examined by qRT-PCR (n=3). (B) Flow cytometry analysis of Chrm1⁺ cells in SMGs. (C) Immunofluorescence staining of Chrm1.

Fig. 6

Effects of IR and transient Hh activation on gene expression in SMGs of female mice and human salivary epithelial cells. (A,B) Gene expression examined by qRT-PCR in SMGs of female C57BL/6 mice 10 or 120 days after IR or in SMGs of female Ptch1-lacZ mice treated with SAG for 3 days (A, n=3), and in human salivary epithelial cells 10 days after IR with or without infection of Gli1 or GFP adenoviruses (B, n = 4). (C) Western blot and relative quantification of human P21 (n = 4).