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

Abstract

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).

Figure 1.

Expression of Hedgehog receptor and downstream molecules in rat meibomian glands. (A) At P3, Smo was broadly expressed in almost all epithelial cells. By 6 weeks, Smo was expressed in all acini, and by 12 weeks, the expression of Smo in acini were generally low. (B) Gli1 was expressed in the cytoplasm of meibomian glands at 3 days, 6 weeks, and 12 weeks old. (C) Gli2 was highly expressed in the cytoplasm at 3 days old, but by 6 weeks and 12 weeks, the expression was almost absent. (D) Gli3 was hardly expressed at 3 days, 6 weeks, and 12 weeks. Du, duct; Ac, acinus.

Figure 2.

Serum and azithromycin promoted rat Meibomian gland epithelial cells differentiation. (A) Cells were small polygons in MGECM, and large and flattened cellular appearances with plenty of bright vesicles appeared in the cytoplasm after 3 days cultivation in DM or DM + AZM. (B, C) Nile red staining showed cytoplasmic lipid accumulation increased in DM, and the addition of AZM promoted lipid accumulation. *P < 0 .05; ***P < 0.001. MGECM, meibomian gland epithelial cells medium; DM, differentiation medium; DM + AZM, DM added with azithromycin.

Figure 3.

Decreased in the expression of Smo and Gli1 accompanied with the increase of differentiation in vitro. (A) The relative expression of Smo protein slightly declined in DM, and significantly decreased in DM + AZM. (B) Western Blot showed the expression of Gli1 had a similar decline as Smo in different media. (C, D) The relative expression of Gli2 and Gli3 proteins did not show a significantly difference in MGECM, DM, and DM + AZM. (E) Immunofluorescence staining of Glis showed that Gli1 was mainly expressed in the nucleus, and the expression of Gli1 in MGECM was higher. Gli3 was mainly expressed in the cytoplasm, and Gli2 was hardly expressed. (*P < 0 .05). MGECM, meibomian gland epithelial cells medium; DM, differentiation medium; DM + AZM, DM added with azithromycin.

Figure 4.

Inhibition of Hedgehog signaling pathway blocked cell proliferation. (A) PCR showed that the expression of Gli1 raised 14.71-fold after exposed to 0.6 µM SAG, and the expression of Gli1 reduced to 0.52-fold after exposed to 15 µM cyclopamine in DM + AZM. The mRNA expression of Ki67 decreased to 0.78-fold in the cyclopamine group. (B) The cell morphology after treated with drugs for 3 days in MGECM. (C) Cell proliferation analysis showed a considerable loss of cell number after treated with 15 µM cyclopamine. (D, E) The immunofluorescence staining showed that the expression of Ki67 was suppressed after treated with cyclopamine in MGECM. *P < 0.05; ***P < 0.001. SAG, MGECM added with 0.6 µM SAG; Cyclopamine, MGECM added with 15 µM cyclopamine.

Figure 5.

Inhibition of Hedgehog signaling pathway promoted cell differentiation, vice versa. (A) The cell morphology after treated with drugs for 3 days in DM + AZM. (B) PCR showed that the expression of Gli1 raised after exposed to 0.6 µM SAG, and decreased after exposed to 15 µM cyclopamine in DM + AZM. The expression of SREBP1 rose in the cyclopamine group and reduced in the SAG group. (C, D) Nile red staining showed an accelerated lipid accumulation after treated with 15 µM cyclopamine and an obvious decline in the SAG group in DM + AZM. *P < 0.05; **P < 0.01; ***P < 0.001. SAG, DM + AZM added with 0.6 µM SAG; Cyclopamine, DM + AZM added with 15 µM cyclopamine.