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).

Single-Cell Ribonucleic Acid Sequencing Clarifies Cold Tolerance Mechanisms in the Pacific White Shrimp (Litopenaeus Vannamei)

“…The “hedgehog signaling pathway” (ko04340), which regulates cell fate, including proliferation, apoptosis, migration, and differentiation ( Qu et al, 2021 ; Wutka et al, 2014 ), was significantly enriched in cell cluster 2. Ccnd2, which was significantly upregulated in cell cluster 2, is a key gene in the “hedgehog signaling” pathway ( Li et al, 2020 ) ( Figure 1E ).…”

Activation of ADRB2/PKA Signaling Pathway Facilitates Lipid Synthesis in Meibocytes, and Beta-Blocker Glaucoma Drug Impedes PKA-Induced Lipid Synthesis by Inhibiting ADRB2

“…However, they evaluated the cornea and conjunctiva, but not the meibomian glands. Qu et al reported the antagonistic role of hedgehog signaling in the proliferation and differentiation of rat MGECs, suggesting that the PKA pathway might play a role in the differentiation of MGECs because the PKA pathway is an antagonist of hedgehog signaling [ 6 ]. In this study, we treated HMGECs with forskolin to increase cAMP levels and evaluated CREB phosphorylation to elucidate the role of the PKA pathway in lipid synthesis in meibocytes.…”

“…The loss of the CD147-mediated pathway was associated with a lower number of acini and a decrease in lipid-filled meibocytes in CD147 knockout mice [ 5 ]. The hedgehog signaling pathway has an antagonistic effect on the proliferation and differentiation of rat meibomian gland epithelial cells [ 6 ]. Transcriptome analysis revealed multiple pathways, such as the insulin signaling pathway, mammalian target of rapamycin pathway, and peroxisome proliferator activator receptor (PPAR) signaling, which are possibly associated with lipid synthesis in HMGECs and mouse meibocytes [ 7 , 8 ].…”

Ductal Hyperkeratinization and Acinar Renewal Abnormality: New Concepts on Pathogenesis of Meibomian Gland Dysfunction

“…Based on our recent finding, DNase2 is a differentiation marker expressed in the central differentiated epithelial cells of the MGs but not in the peripheral undifferentiated cells [ 14 ]. It has been demonstrated that the self-renewal of acinar cells might be regulated by the Hedgehog pathway [ 16 ], which is a family of secreted proteins. It plays a role in regulating the genes of transcription involved in proliferation and differentiation [ 37 ].…”

“…The abnormal proliferation and differentiation of meibocytes are the main causes of MGD rather than the hyperkeratinization of the ductal system [ 9 , 10 , 13 ]. Recent studies from our laboratory suggested that the co-effects of the hyperkeratinization of the ducts and the abnormal self-renewal of the acinar cells lead to MGD [ 14 , 15 , 16 ]. In addition, bacteria have a critical role in the occurrence of MGD [ 17 , 18 , 19 ].…”