Hedgehog Signaling Pathway Regulates the Proliferation and Differentiation of Rat Meibomian Gland Epithelial Cells

Purpose: Meibomian glands play a vital role in maintaining ocular surface stability. This study aimed to investigate whether Hedgehog signaling is involved in the regulation of meibomian gland epithelial cells.

Methods: Rat meibomian glands epithelial cells (RMGECs) were isolated from ducts and ductules, and then were cultivated to passage two on Matrigel coated wells in meibomian gland epithelial cells medium (MGECM). Cells were switched from MGECM to differentiation medium (DM) or DM added 10 µg/mL azithromycin (DM + AZM) when reached 50% to 60% confluence. The effects of the Smoothened (Smo) agonist (Smo agonist [SAG]) and antagonist (by cyclopamine) on RMGECs were analyzed using quantitative RT-PCR, cell proliferation analysis, immunofluorescence staining, and Nile red staining.

Results: The Hedgehog receptor, Smo, and its downstream molecules, Glis, were expressed both in vivo and in vitro. Smo and Gli1 both decreased with the increase of differentiation in vitro. Smo antagonist, cyclopamine, reduced cell numbers, and the expression of Ki67 in MGECM, and promoted the expression of SREBP1 and lipid production in DM + AZM. Smo agonist, SAG, inhibited the expression of SREBP1 and lipid accumulation in DM + AZM but showed no significant effects on raising cell numbers and the expression of Ki67 in MGECM.

Conclusions: The Hedgehog signaling pathway appears to play important roles in RMGECs proliferation and differentiation. This may provide a potential therapeutic way to treat meibomian gland dysfunction (MGD).