Effects of ductal ligation/de-ligation on mTOR pathway and morphology of salispheres

Objectives: Cultured salivary gland stem/progenitor cells form spherical, floating clusters in vitro named as salispheres. Salispheres usually develop around day two to day three of culture and express different stem cell markers such as; c-kit. Ductal ligation of submandibular gland (SMG) induces atrophy where by cells undergo apoptosis and fibrosis while de-ligation results in the recovery of the gland. The mammalian target of rapamycin (mTOR) is one of the signaling pathways that plays a role in cell growth and proliferation. It consists of two main complexes; mTORC1 and mTORC2. The activation of mTORC1 leads to the phosphorylation of the two main substrates; 4e-bp1 and S6K. Since mTOR is active during healthy salispheres development, the aim of this study was to measure mTOR activity in ligated/de-ligated models and evaluate the morphological changes in salispheres.
Methods: The SMG of female, ICR mice were used in this study. Duct ligation of SMG was performed for 7 days whereas de-ligation was processed for additional 7 days. All glands were collected and digested with collagenase II and hyaluronidase, washed, filtered and plated in 12-wells dish. Salispheres were then imaged by the phase-contrast microscopy and collected for analysis using western blot and immunofluorescence.
Results: Results showed that number of salispheres was significantly lower in ligated glands when compared to un-operated glands and to de-ligated glands. In addition, salispheres from ligated glands showed mTOR activity and both ligation and de-ligation cause morphological changes to salispheres. These results suggest that the morphological changes caused by ligation/de-ligation are not mTOR related but instead Rho-associated protein kinase (ROCK) maybe a reason for these morphological changes.
Conclusions: In conclusion, stem/progenitor cells from atrophic/regenerated glands are in a different state compared to stem/progenitor cells from normal tissues.