Figure 1.

SHH Actively Secreted in Response to Various Injury Signals

SHH is considered a morphogen that regulates pattern formation during embryonic limb development. In addition to this canonical function, we hypothesized that SHH is selectively secreted under specific cell-damaging conditions as a stem cell-activating factor (A). Endometrial stem cells were incubated in standard culture medium with or without H₂O₂ (10 mM) for 30 min, after which the medium was replaced with serum-free medium, and the cells were cultured for 48 h (B). Endometrial stem cells were exposed to acute X-ray radiation at a dose of 4 Gy, after which the medium was replaced with serum-free medium, and the cells were cultured for 48 h (C). Endometrial stem cells were cultured with or without serum for 48 h (D). The 2% TCA treatment (150 μL, administered directly into uterine horn) caused significant histological uterine endometrial ablation. The acute TCA treatment caused significant histological endometrial damage with increased degenerative vacuoles and apoptosis (E). After albumin/immunoglobulin depletion, the proteins in the serum samples were precipitated with 10% TCA and subjected to SDS-PAGE. To prove that media samples are not contaminated with cytosolic or nuclear content during the TCA precipitation procedure, the levels of actin in the culture medium were analyzed. Compared with the uninjured control mice, TCA-induced acute endometrial ablation resulted in a significant increase in SHH secretion into the peripheral circulation (F). β-Actin was used as the internal control. The results are presented as the mean ± SEM from three independent experiments.