Y-27632-mediated long-term expansion of salivary gland epithelial cells

Abstract In salivary glands, epithelial cells perform functional activities such as the production and secretion of saliva. Therefore, the use of salivary gland epithelial cells to study general biology or drug screening may contribute to the development of therapeutic strategies for salivary gland diseases. However, the underlying mechanism of long-term in vitro expansion methods for salivary gland epithelial cells (SG-Epis) are unknown. Here, we present the roles of the rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms. The treatment with Y-27632 significantly increased the proliferative potential and maintained the expression of Krt8 and Krt14 for 17 passages. Conversely, in the absence of Y-27632, SG-Epis lost their epithelial morphology. The treatment with Y-27632, however, maintained the epithelial morphology and downregulated the mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 reveals that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis. RNA interference study shows that ROCK2/JNK signaling is also required for proliferation and maintenance of SG-Epis. The culture method using Y-27632 provides a solution for efficient in vitro expansion of SG-Epis.