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).

Meibomian glands are the large specialized sebaceous glands in the human body.¹ The acini of meibomian glands synthesize meibum and secrete meibum through holocrine secretion. The meibum is continuously transported to the ocular surface through ductules and ducts, becoming the main source of the lipid layer for the tear film. The lipid layer has vital functions that slows the evaporation of the aqueous layer of the tear film, preserves the optical surface clear, and forms a barrier to protect the eye.² Acinar atrophy that leads to a low secretion contributes to meibomian gland dysfunction (MGD).¹ MGD is widely prevalent worldwide ranging from 3.5% to almost 70%, and the rate appears to be especially higher in Asian populations.³ However, there is no cure for MGD, partly because the biological behaviors of meibomian glands, such as proliferation and differentiation, remain unclear.⁴ 

The Hedgehog signaling pathway is known to play essential roles in many cell fate decisions, including polarity, differentiation, and proliferation.⁵ The Hedgehog signaling pathway also regulates the proliferation and differentiation of sebaceous glands.^6–8 The Hedgehog is a family of secreted proteins. Once Hedgehog binds to the Patched (Ptch), a transmembrane protein locating in the cell membrane, then Smoothened (Smo), another transmembrane protein locating in the cell membrane, is released from Ptch. Next, the downstream molecule Zinc finger family transcription factors, Glis, which have three types Gli1, Gli2, and Gli3, are released from the complex. Subsequently, Glis translocates into the nucleus and regulates the transcription of genes involved in proliferation, differentiation, and so on.^9,10 Although the Hedgehog signaling pathway is involved in many tissue regulations,^11,12 its role in meibomian glands is unknown. 

In this study, we investigated the activation and inhibition of Smo in rat meibomian gland epithelial cells (RMGECs). We sought to determine whether and how the Hedgehog signaling pathway is involved in meibomian gland epithelial proliferation and differentiation. 

Materials and Methods

Animals

Male Sprague-Dawley rats were purchased from Laboratory Animal Center of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China). Male Sprague-Dawley rats about 6 weeks old, weighing 200 to 220 g, were the origin of primary cells in this study. Animals were studied in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (IACUC number S2305). 

Culture of Primary Rat Meibomian Gland Epithelial Cells

After rats were euthanized, meibomian gland tissues were isolated under a dissecting microscope by removing the skin, connective tissue, muscle, palpebral conjunctiva, and eyelashes. Tissues were cut into small pieces each containing 3 meibomian glands, and further digested with 2.5 mg/mL collagenase I (Biosharp, Anhui, People's Republic of China) at 37°C for 2.5 hours. Acini were separated from ducts using microsurgical forceps in a petri dish (Supplementary Figs. S1A, S1B). Ducts and connective tissues were discarded. The remains in the dish were collected and gently blown with a pipette, followed by filtration through nylon mesh with 100-micron pores. The filtrate was centrifuged at 1200 r/min for 5 minutes, and the cells were resuspended in meibomian gland epithelial cells medium (MGECM) and seeded in 6-well plates coated with The Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA).^13,14 MGECM was a serum-free medium modified from Dongfang Yu¹⁵ in which cells proliferate fast, consisting of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, [1:1]; Hyclone, Logan, UT, USA), 100 U/mL Penicillin, 0.1 mg/mL Streptomycin (Solarbio, Beijing, People's Republic of China), 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL sodium selenite (Gibco, Grand Island, NY, USA), 8.4 ng/mL cholera toxin (Sigma-Aldrich Corp., St. Louis, MO, USA), 10 ng/mL epidermal growth factor (Peprotech, Rocky Hill, NJ, USA), 0.4 µg/mL hydrocortisone (Sigma-Aldrich Corp.), 0.25 µg/mL amphotericin B (Invitrogen, Grand Island, NY, USA), and 10 µmol/L Y-27632 (Enzo Life Sciences, Farmingdale, NY, USA).^15,16 Y-27632 is a ROCK inhibitor promoting epithelial cell proliferation.¹⁷ Cells were cultivated at 37°C in a humidified incubator with 95% air and 5% CO₂. Medium was changed every 2 days. 

Treat Cells With Different Culture Conditions

When cells reached about 90% confluence, cells were digested with Accutase (Sigma-Aldrich Corp.) at 37°C for 30 minutes to subculture. The cultured RMGECs were transferred to the P2 generation. To promote RMGECs differentiation, cells were cultured in a serum containing differentiation medium (DM), consisting of DMEM/F12 added with 10% fetal bovine serum (ScienCell, Carlsbad, CA, USA),¹⁸ or DM added with10 µg/mL of azithromycin (AZM; MedChemExpress, Monmouth Junction, NJ, USA; DM + AZM)¹⁹ to accelerate cell differentiation for 3 days when reaching 50% to 60% confluence. 

Inhibition and Induction of the Hedgehog Signaling Pathway

To inhibit the Hedgehog signaling pathway, RMGECs were treated with 15 µM cyclopamine (Selleck Chemicals, Houston, TX, USA) when reaching 50% to 60% confluence in MGECM or DM + AZM. To active Hedgehog signaling pathway, RMGECs were treated with MGECM or DM + AZM supplemented 0.6 µM Smo agonist (SAG; Cayman Chemical, Ann Arbor, MI, USA). Control groups were treated with medium added vehicle (ethanol, concentration 0.03%). After 3 days of treatments, cells were harvested. 

Immunofluorescence Staining

Fresh rat meibomian gland tissue sections were fixed with 4% paraformaldehyde for 1 hour at room temperature. The sections were dehydrated, embedded in optimum cutting temperature (OCT) compound, and cut into 10 µm thickness sections. After permeated in 0.5% Triton X-100 for 20 minutes, and blocked in goat serum for 30 minutes at room temperature, sections were incubated with primary antibody (Smo, Sc-166685, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA; Gli1, DF7523, 1:100; Affinity Biosciences, Cincinnati, OH, USA; Gli2, Sc-271786, 1:100, Santa Cruz Biotechnology; Gli3, Sc-74478, 1:100, Santa Cruz Biotechnology; Ki67, Ab92742, 1:300, Abcam, Cambridge, MA, USA) at 4°C overnight. A secondary fluorescent antibody (rabbit anti-goat immunoglobulin G [IgG], BA1032, 1:100, Bioster, Wuhan, China) was used for detection. Then, 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich Corp.) was used for nuclear counterstaining. Slides were mounted with antifluorescence quenching agents and imaged under a laser scanning confocal microscope (Nikon A1). 

To analyze the Ki67 positive rate, the labeling index was performed. Three random images from every cell slide were photographed in triplicate. The labeling index was calculated by dividing the number of Ki67 positive cell nuclei by the total number of cells and multiplying by 100.²⁰ 

Nile Red Staining

Nile red is a lipophilic dye. Stock solutions of Nile red (Sigma-Aldrich Corp.) were prepared at 10 mg/mL in dimethyl sulfoxide (DMSO) and stored protected from light. Cell slides were fixed with 4% paraformaldehyde for 20 minutes at room temperature. After wash, fixed cells were incubated within 20 µg/mL Nile red in PBS for 2 to 3 hours at room temperature in the dark. The slides then were washed with PBS, mounted with 2 µg/mL DAPI (Sigma-Aldrich Corp.) for 5 minutes at room temperature. Cells were imaged with a confocal microscope, under the same parameters except for changing specimens and moving view fields. Nine random images from every cell slide were photographed in triplicate. To eliminate the bias of integrated optical density in images caused by cell density differences after treatment, the average fluorescence intensities of images were measured with Image J software.¹⁵ 

Cell Proliferation Analysis

To detect the effects of agonist and antagonist on RMGECs proliferation, cells were seeded into 24-well plates at a density of 1 × 10⁴/well. When cells reached 40% to 50% confluence, the drugs and vehicles were added. After another 3 days cultivation, cells were digested by Accutase and counted using a hemocytometer.^21–23 

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was isolated using TRIzol reagent and Ultrapure RNA Kit (CWBIO; China). After concentration was measured, RNA was reverse transcribed to cDNA using PrimeScript RT Master Mix (Takara, Japan). Subsequently, PCR was performed using TB Green Premix Ex Taq (Takara) in a total volume of 10 µL using amplification condition of at 95°C for 5 min, followed by 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. β-actin was used as a reference gene. The primers sequence pairs used in this study are shown in Supplementary Table S1. Results were analyzed by 2^−∆∆CT analysis. 

Western Blot

Proteins in cells were extracted using RIPA lysis buffer (P0013B, Beyotime Biotechnology, China). The concentrations of proteins were determined. Proteins were put in boiling water for 10 minutes, separated by electrophoresis on 12% SDS-PAGE gels, and transferred to PVDF membranes. The PVDF membranes were blocked by 5% fat-free milk for 2 hours at room temperature, and then sequentially incubated with primary antibody (Smo, Sc-13943, 1:200, Santa Cruz Biotechnology; Gli1, DF7523, 1:1000, Affinity Biosciences; Gli2, Sc-271786, 1:300, Santa Cruz Biotechnology; Gli3, Sc-74478, 1:300, Santa Cruz Biotechnology) and HRP-conjugated goat anti-rabbit IgG antibody (1:5000, BA1054, Boster, China) or goat anti-mouse IgG antibody (1:5000, BA1051, Boster). The proteins were visualized with Super ECL Plus (P1050, Applygen, China). 

Statistics

Data were shown as \(\bar x\) ± standard deviation (SD). One-way analysis of variance (ANOVA) with post hoc LSD test or Dunnett's t-test for multiple groups were performed using SSPS version 22.0 software to analyze data. Each experiment was performed under the same conditions and repeated at least three times. P < 0.05 was considered statistically significant. 

Results

Expression of the Hedgehog Receptor and Downstream Molecules in Rat Meibomian Glands

To clarify the expression patterns of the Hedgehog signaling pathway molecules in meibomian glands in vivo, immunofluorescence staining of Smo, the key signaling transducer,²⁴ and its downstream transcription factors, Gli1, Gli2, and Gli3, were performed in meibomian glands of rats at 3 days, 6 weeks, and 12 weeks old. Three days after birth, perpendicular to the fused eyelid margin, meibomian glands developed to be cords of epithelium, and Smo was broadly expressed in almost all epithelial cells (Fig. 1A). At 6 weeks after birth, the upper and lower eyelid margins had already separated. Ducts and ductules of meibomian glands with clear lumens surrounded by grape-like clusters of acini had already developed, and all acini expressed Smo (see Fig. 1A). However, by 12 weeks, the expression of Smo in acini was generally low (see Fig. 1A). Gli1 was expressed in the cytoplasm of meibomian glands at 3 days, 6 weeks, and 12 weeks old (Fig. 1B). Gli2 was highly expressed in the cytoplasm at 3 days old, but by 6 weeks and 12 weeks, the expression was almost absent (Fig. 1C). Gli3 was hardly expressed at 3 days, 6 weeks, and 12 weeks (Fig. 1D). 

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.

By performing immunofluorescence staining of Smo and Glis, we confirmed that Smo, Gli1, and Gli2 were expressed in rats after birth, but Gli3 was not. As transcription factors, Glis translocates from the cytoplasm to the nucleus to play their roles when the Hedgehog signaling pathway is activated.²⁵ The expression of Gli1 and Gli2 both appeared in the cytoplasm, indicating that although molecules of the Hedgehog signaling pathway were expressed in vivo, the signaling pathway may be quiescent in postnatal rats. 

Serum and Azithromycin Promoted Rat Meibomian Gland Epithelial Cells Differentiation

To explore the role of the Hedgehog signaling pathway in vitro, cells originated from meibomian glands of rats at 6 weeks were cultured. MGECM was used as a proliferation medium in this study. In MGECM, cells proliferated rapidly in polygonal shapes. After the medium switching from MGECM to DM or DM + AZM for another 3 days, the morphology of cells was changed to large and flattened cellular appearances^15,26 with plenty of bright vesicles appearing in the cytoplasm (Fig. 2A). Nile red staining, a specific staining for neutral lipid,²⁷ showed cytoplasmic lipid accumulation increased in DM, and the addition of AZM promoted lipid accumulation (P < 0.05 for both, n = 3 in each group; Figs. 2B, 2C). Serum and AZM can promote lipid accumulation in meibocytes.^18,19 Lipid secretion is an important character of the maturation of meibocytes.²⁸ Therefore, we confirmed the cells we cultured were meibomian gland epithelial cells, and proliferation and differentiation models of rat meibomian gland epithelial cells in vitro were established. 

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.

Decreased in the Expression of Smo and Gli1 Accompanied With the Increase of Differentiation In Vitro

Because the effects of DM + AZM on promoting cell differentiation were stronger than DM, DM + AZM was selected for differentiation models in vitro in the following studies. To verify whether Smo is expressed in vitro, Western blot analyses were performed. The relative expression of Smo protein was lower to 0.88-fold in DM (P > 0.05) and 0.59-fold in DM + AZM (P < 0.05) compared to MGECM (n = 3 in each group). Western blot analyses showed a slight decline of Smo protein in DM and a significant reduction in DM + AZM (Fig. 3A). 

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.

The expressions of the downstream molecules, Glis, were also detected. Western Blot analyses showed a similar decline as Smo in the expression of Gli1. The relative expression of Gli1 protein was slightly lower to 0.85-fold in DM (P > 0.05) and significantly lower to 0.41-fold in DM + AZM (P < 0.001) compared to MGECM (n = 3 in each group; Fig. 3B). The expression of Gli2 and Gli3 didnot show a significant difference in MGECM, DM, and DM + AZM (P > 0.05, n = 3 in each group; Figs. 3C, 3D). Immunofluorescence staining of Glis (Fig. 3E) showed that Gli1 was mainly expressed in the nucleus, Gli3 was mainly expressed in the cytoplasm, and Gli2 was hardly expressed. Besides, compared with the expression levels in DM and DM + AZM, the expression of Gli1 in MGECM was higher, which was the same as the result of Western blot analyses. 

This part of the result demonstrated that the Hedgehog receptor and downstream molecules were also expressed in vitro. Gli1, which was originally expressed in the cytoplasm in vivo at 6 weeks old, was translocated to the nucleus after cultivation in vitro. Therefore, the quiescent Hedgehog signaling pathway in vivo was activated in vitro. Moreover, both the expression of Smo and Gli1 expression decreased with the increase of differentiation, indicated that the Hedgehog signaling pathway may be related to the proliferation and differentiation of meibomian glands. 

Inhibition of the Hedgehog Signaling Pathway Blocked Cell Proliferation

To investigate how the Hedgehog pathway affects cell proliferation, we added the Smo agonist, SAG, or a potent antagonist, cyclopamine, to MGECM. Induction and inhibition of Smo were confirmed by the increase and reduction of the mRNA expression of Gli1 (Fig. 4A), the major downstream transcription factor. The expression of Gli1 mRNA rose to 14.71-fold induced by 0.6 µM SAG (P < 0.001) and reduced to 0.52-fold by 15 µM cyclopamine (P < 0.05; n = 6 in each group). Cell numbers in control groups, SAG groups, and cyclopamine groups were 30.71 ± 4.98 × 10⁴/well, 29.42 ± 1.38 × 10⁴/well, and 4.21 ± 1.30 × 10⁴/well (n = 3 in each group), respectively (Figs. 4B, 4C) after culturing for 3 days. Cell proliferation analysis showed a considerable loss of cell numbers after treated with 15 µM cyclopamine in MGECM (P < 0.05). However, SAG had no obvious effect on increasing cell numbers (P > 0.05). 

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.

To further investigate the roles of Smo in RMGECs proliferation, PCR and immunofluorescence staining were performed to detect the expression of Ki67, a marker of cell proliferation.²⁰ The relative mRNA expression of Ki67 was increased to 1.11-fold after exposed to SAG (P > 0.05) and decreased to 0.78-fold by cyclopamine (P < 0.05) compared to the control group (n = 4 in each group; see Fig. 4A). Immunofluorescence staining showed that the labeling index of Ki67 in the SAG group slightly raised to 9.02 ± 4.56 compared to 7.19 ± 2.25 in the control group, but the difference was of no significance (P = 0.64, n = 3 in each group). Although the labeling index of Ki67 in the cyclopamine group markedly reduced to 1.42 ± 0.83 (P < 0.001; Figs. 4D, 4E). Collectively, we demonstrated that inhibition of Smo led to a reduction in proliferation of RMGECs. 

Inhibition of the Hedgehog Signaling Pathway Promoted Cell Differentiation

To study how the Hedgehog pathway influences cell differentiation, cells were exposed to SAG and cyclopamine in DM + AZM (Fig. 5A). Induction and inhibition of Smo were confirmed by the upregulation and downregulation of Gli1 mRNA expression after treated with SAG and cyclopamine for 3 days (n = 5 in each group; Fig. 5B). To examine the lipid production in RMGECs, we choose sterol regulatory element-binding protein 1 (SREBP1), a key regulator of lipid synthesis,²⁹ as a differentiation marker. PCR (see Fig. 5B) showed the expression level of SREBP1 was induced to 1.26-fold after treated with cyclopamine for 3 days (P = 0.001). Conversely, it reduced to 0.86-fold after treated with SAG (P < 0.05; n = 4 in each group). Nile red staining was also performed and the average fluorescence intensity of images was analyzed. Nile red staining showed an accelerated lipid accumulation after treated with cyclopamine and an obvious decline in the SAG group (Figs. 5C, 5D; P < 0.01 for both, n = 3 in each group). We also examined the SREBP1 mRNA expression (n = 4 in each group) and Nile red staining (n = 3 in each group) in MGECM after being treated with 0.6 µM SAG and 15 µM cyclopamine. The results were similar to those in DM + AZM (Supplementary Fig. S2). Accordingly, we demonstrated that inhibition of Smo promoted cellular differentiation, meanwhile induction of Smo reduced cellular differentiation. 

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.

Discussion

The most common pathogenesis of MGD is ductal epithelial hyperkeratinization^28,30 and acinar atrophy.³¹ Here, we focused on the role of the Hedgehog signaling pathway in the proliferation and differentiation of acini attempting to provide a new target for MGD treatments. In this study, we demonstrated that the Hedgehog receptor, Smo, and its downstream molecules, Glis, were expressed both in vivo and in vitro. The expression of Smo and Gli1 decreased with the increase of differentiation in vitro. Our findings showed that inhibition of Smo restrained cell proliferation and induced lipid accumulation, and induction of Smo suppressed lipid production but had no significant effect on cell proliferation. Overall, our study first directly demonstrated that the Hedgehog signaling pathway regulates the proliferation and differentiation of RMGECs. 

Previous researchers have established several culture systems for meibomian gland epithelial cells derived from the rabbit, rat, mouse, and human, however, they often take the method of digesting the entire meibomian gland,^{13,15,16,27,28} which makes the acinar cells mixed with ductal cells. In our study, rat acinar cells were manually separated from ducts, and ducts were discarded rendering the acinar cells we cultured purer. Due to the scarcity of human meibomian glands, we used RMGECS instead of human cells. 

Accumulated researches have shown that the Hedgehog signaling pathway is in the regulation of sebaceous glands. SZ95 sebocytes, a sebaceous lineage, proliferation decreased and differentiation increased after treated with an antagonist of the Hedgehog signaling pathway.⁶ In transgenic mice, enhanced Hedgehog signaling correlated with increased size and number of sebaceous glands,^7,8 enlargement and elongation of sebaceous gland ducts,⁷ and formation promotion of sebaceous glands. However, the role of the Hedgehog signaling pathway in the regulation of meibomian glands is not known. 

Our studies demonstrated that the key molecule of the Hedgehog signaling pathway, Smo, was expressed both in vivo and in vitro, but its downstream molecules Glis differed in expression. Glis were Zinc finger family transcription factors, which have 3 types, Gli1, Gli2, and Gli3. Different transcription factors are involved in different processes. Gli1 and Gli2 mostly serve as positive feedbacks to Hedgehog, whereas Gli3 functions as a repressor.³² In our study, Gli1 was expressed at 3 days, 6 weeks, and 12 weeks old of rats in vivo, Gli2 was expressed at 3 days but was absent at 6 weeks and 12 weeks, and Gli3 was hardly expressed at these ages. After cultivation in vitro, Gli1 translocated to the nucleus, indicating it was activated, whereas Gli3 was mainly expressed in the cytoplasm, and Gli2 was almost absent. The differences in the expression of Glis indicate that the Hedgehog signaling pathway in the meibomian glands passes the signal downstream may mainly through Gli1 rather than Gli2 and Gli3. 

SAG and cyclopamine, both are synthetic small molecules, respectively induce and inhibit the Hedgehog signaling pathway by binding to the Smo heptahelical bundle and differently changing the conformation of the Smo protein.³³ Therefore, we selected the expression level of the downstream molecule, Glis, to evaluate whether Smo was induced or inhibited. PCR showed immense changes in the expression level of Gli1 after treatments with agonist and antagonist, whereas Gli2 and Gli3 expressed at a relatively low level with small changes (see Supplementary Fig. S3). The result further supported that Gli1 was the major transcript factor of the Hedgehog signaling pathway in the meibomian glands. 

The proliferation of RMGECs in MGECM and differentiation in DM + AZM were studied. The same initial cell density of SAG and control groups as cyclopamine was used at the beginning of our cell proliferation analysis in MGECM. However, cells in the SAG and control groups in MGECM proliferated so fast that the cells often overgrew the entire well just 2 days after the drug treatment. After reaching contact inhibition, the cells stopped proliferating. To eliminate cell proliferation analysis errors caused by cell contact inhibition, the initial seeding density of the SAG group and the control group was reduced and the drug was added at a lower cell confluence. Cells did not grow over the entire well after 3 days of treatment, but the results still showed that SAG did not significantly increase cell number (data not shown). Besides, we examined the expression of proliferation marker, Ki67, through PCR and immunofluorescence staining that further verified that SAG did not significantly promote cell proliferation. On the other hand, after exposed to SAG, the lipid production and the expression level of differentiation marker, SREBP1, obviously decreased in DM + AZM. The results demonstrated that although induction of Smo had no obvious effects on cell proliferation, it inhibited cell differentiation. Conversely, our results showed that treatment of RMGECs with the antagonist of Smo, cyclopamine, decreased proliferation in MGECM and stimulated differentiation in DM + AZM, similar to the results of C. Niemann et al. in SZ95 sebocytes.⁶ Besides, even in MGECM, cyclopamine still worked effectively as a promotor of cell differentiation. 

Our experimental results are similar to a study of Werminghaus et al.³⁴ that SAG has no obvious promotion effect on proliferation but cyclopamine results in an inhibitory effect on the proliferation of adrenocortical cells. In their study, they mentioned the possible reason may be the cross effects between cAMP-PKA pathway and the Hedgehog signaling pathway. The cAMP-PKA pathway is a strong antagonist of Hedgehog signaling. SAG may not be able to release the antagonism that inhibits the effects of the cAMP-PKA pathway on Hedgehog signaling, whereas cyclopamine enlarges the PKA mediated inhibition on Hedgehog signaling. In our study, Gli1 in MGECM was raised to 14.71-fold, but it showed no effect on cell proliferation. The cross action of the cAMP-PKA pathway on the Hedgehog signaling pathway may also work on RMGECs. 

In conclusion, our results demonstrated that the molecules of the Hedgehog signaling pathway were expressed in rats both in vivo and in vitro. Although the signaling pathway was quiescent in vivo, it was activated in vitro. Through the in vitro culture models, we proved that the Hedgehog signaling pathway was involved in the regulation of meibomian glands. Manipulating the Hedgehog signaling pathway may help build the meibomian glands tissue engineering in the future in vitro. Smo may also be a useful new target to treat MGD. Further animal experiments and clinical trials are needed to help figure out the therapeutic effects of Smo on MGD. 

Acknowledgments

Supported by National Key Research and Development Program of China (2017YFE0103500), National Natural Science Foundation of China (82070934), the Fundamental Research Funds for the Central Universities (HUST: 2019kfyXMBZ065) and Research Foundation of Union Hospital (2018xhyn106). 

Author Contributions: J.-Y.Q.: Collection and assembly of data, data analysis and interpretation, and manuscript writing; Y.-T.X.: Collection and assembly of data, data analysis, and interpretation; Y.-Y.Z.: Collection and assembly of data, data analysis, and revised manuscript; H.-T.X.: Conception and design, collection and assembly of data, data analysis and interpretation, and final approval of manuscript; M.-C.Z.: Conception and design, data analysis and interpretation, and final approval of manuscript. 

Disclosure: J.-Y. Qu, None; Y.-T. Xiao, None; Y.-Y. Zhang, None; H.-T. Xie, None; M.-C. Zhang, None