Abstract
Purpose.:
We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses.
Methods.:
Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures.
Results.:
Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells.
Conclusions.:
Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.
Meibomian gland secretion is critically dependent upon the active proliferation of glandular epithelial cells.
1 Once generated, these sebaceous-like cells undergo a maturation process toward terminal differentiation, lipid production, and holocrine secretion. Such secretion involves the death and disintegration of fully mature, lipid-rich epithelial cells, their release into glandular ductules, and ultimately, delivery to the ocular surface.
1,2 Given this continual loss of cells, stimulation of epithelial cell proliferation is extremely important and promotes not only meibocyte replenishment, but also the production of meibum.
The stimuli that induce proliferation of human meibomian gland epithelial cells are unknown. We hypothesize that epidermal growth factor (EGF), as well as pituitary hormones and growth factors (i.e., in the components of bovine pituitary extract [BPE]) will induce such proliferation, but not differentiation (e.g., lipid accumulation). We also hypothesize that these actions will involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation.
In support of our hypotheses, researchers have shown that EGF is a strong mitogen that stimulates the clonal growth of rabbit meibomian gland epithelial cells,
3 enhances the mitotic activity of hamster sebocytes,
4,5 and promotes the proliferation, but not differentiation, of human amnion epithelial cells by regulating cell cycle genes.
6 Similarly, several pituitary hormones increase sebaceous gland epithelial cell growth,
2 and BPE augments the proliferation of human mammary, thymic, ureteral, and conjunctival epithelial cells.
7–10 Scientists also have reported that EGF and BPE increase the proliferation of normal and immortalized keratinocytes, whereas factors, such as serum, induce their terminal differentiation.
11 In addition, hamster sebocytes exposed to EGF do not accumulate intracellular lipids.
5
The purpose of our study was to test our hypotheses with human meibomian gland epithelial cells. Toward that end, we examined the effects of EGF, BPE, and both factors in combination, on the proliferation, lipid accumulation, and gene expression of these cells in vitro.
Immortalized human meibomian gland and conjunctival epithelial cells (gift of Ilene K. Gipson, Boston, MA) were cultured on glass coverslips (Electron Microscopy Sciences, Hatfield, PA) placed in 6-well plates in SFM supplemented with 5 ng/mL EGF and either 50 μg/mL BPE (meibomian cells) or 25 μg/mL BPE (conjunctival cells). When cells reached approximately 75% confluence, medium was changed to SFM, SFM supplemented with 50 μg/mL BPE and 5 ng/mL EGF, or serum-containing medium. Media were replaced every two to three days for seven days, when cells were washed, then fixed in 4% paraformaldehyde for 30 minutes. Following additional washes, coverslips were exposed to LipidTOX Green neutral lipid stain (Invitrogen, Grand Island, NY) in a humid chamber for 30 minutes. Coverslips were mounted on slides with ProLong Gold antifade reagent with DAPI (Invitrogen) and allowed to dry overnight before imaging with a Nikon Eclipse E800 (Nikon Instruments, Melville, NY).
The differential expression of selected genes was confirmed by using quantitative real-time PCR (qPCR) methods. The cDNAs were transcribed by using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers (Invitrogen). The qPCR reactions were performed in triplicate by using TaqMan Gene Assays (Applied Biosystems, Inc., Foster City, CA), TaqMan-specific primers and probes for small proline-rich protein 3 (Hs01878180_s1), keratin 10 (Hs00166289_m1), TIMP metallopeptidase inhibitor 1 (Hs00171558_m1), laminin alpha 3 (Hs00165042_m1), and GAPDH (4326317E). Differential gene expression was calculated by following the Comparative Ct method, as described in Applied Biosystems User Bulletin 2 (updated 2001).
Effect of EGF and BPE on the Proliferation of Human Meibomian Gland Epithelial Cells
Influence of EGF and BPE on Gene Expression in Human Meibomian Gland Epithelial Cells
To examine whether growth factors upregulate genes linked to cell cycle processes, and downregulate genes associated with differentiation, we treated human meibomian gland epithelial cells with EGF and/or BPE for varying periods of time and then processed the cells for analyses with Illumina BeadChips and bioinformatic software.
In the first set of experiments, we cultured subconfluent, passage 16, human meibomian gland epithelial cells for 2 days in SFM (
n = 3.76 × 10
4cells/well), followed by an additional 5 days in media containing EGF, BPE, or EGF + BPE (
n = 3 wells/condition). Our findings demonstrate that growth factor-induced proliferation is associated with a significant (
P < 0.05) change in the expression of thousands of genes (
Table 1). As compared to the basal state, EGF, BPE, and their combination stimulated a significant increase in the expression of genes associated with the cell cycle, cell proliferation, DNA replication, ribosomes, and translation (
Table 2). Conversely, these growth factors caused a significant decrease in ontologies related to cell differentiation, keratinization, tissue development, and fatty acid and lipid metabolic processes (
Table 3).
Table 1. Effect of EGF and/or BPE on Gene Expression in Human Meibomian Gland Epithelial Cells
Table 1. Effect of EGF and/or BPE on Gene Expression in Human Meibomian Gland Epithelial Cells
Group | Genes ↑ | Genes ↓ | Total Genes |
EGF | 1711 | 1709 | 3420 |
BPE | 1941 | 1803 | 3744 |
EGF + BPE | 1622 | 1670 | 3292 |
Table 2. Growth Factor Upregulation of Cell Cycle and Translational Process Ontologies
Table 2. Growth Factor Upregulation of Cell Cycle and Translational Process Ontologies
Ontology | EGF Genes ↑ | EGF Z-Score | BPE Genes ↑ | BPE Z-Score | EGF + BPE Genes ↑ | EGF + BPE Z-Score |
Cell cycle |
Cell cycle | 147 | 5.9 | 231 | 12.9 | 167 | 8.8 |
Cell cycle arrest | 54 | 5.5 | 75 | 8.4 | 60 | 7.1 |
Cell cycle checkpoint | 41 | 5.9 | 65 | 10.6 | 51 | 8.8 |
Cell cycle phase | 95 | 5.1 | 169 | 13.4 | 123 | 9.6 |
Cell cycle process | 112 | 5.3 | 190 | 13.0 | 138 | 9.2 |
Cell division | 52 | 3.9 | 92 | 9.9 | 70 | 7.7 |
Cell growth | | | 38 | 2.2 | 33 | 2.3 |
Cell proliferation | 143 | 5.1 | 155 | 4.5 | 131 | 4.3 |
Chromosome | | | 98 | 7.8 | 70 | 4.9 |
Chromosome, centromeric region | | | 42 | 8.6 | 29 | 5.7 |
Condensed chromosome | 19 | 2.4 | 46 | 9.8 | 30 | 6.0 |
Condensed chromosome kinetochore | 11 | 2.2 | 27 | 8.3 | 18 | 5.4 |
Condensed chromosome, centromeric region | 12 | 2.4 | 29 | 8.6 | 20 | 5.9 |
DNA replication | 34 | 3.4 | 57 | 7.7 | 44 | 6.1 |
G1/S transition checkpoint | 12 | 2.3 | 14 | 2.6 | 12 | 2.5 |
G1/S transition of mitotic cell cycle | 31 | 4.8 | 43 | 7.1 | 35 | 6.2 |
G2/M transition of mitotic cell cycle | 11 | 0.5 | 25 | 4.4 | 20 | 3.7 |
Interphase | 51 | 4.7 | 80 | 9.1 | 63 | 7.5 |
Interphase of mitotic cell cycle | 50 | 4.7 | 77 | 8.8 | 61 | 7.3 |
Kinetochore | | | 29 | 7.9 | 20 | 5.3 |
M phase | 56 | 3.3 | 112 | 11.4 | 79 | 7.8 |
M phase of mitotic cell cycle | 43 | 3.3 | 93 | 11.8 | 65 | 8.1 |
M/G1 transition of mitotic cell cycle | 14 | 3.1 | 26 | 7.2 | 19 | 5.4 |
Mitosis | 42 | 3.3 | 90 | 11.6 | 64 | 8.2 |
Mitotic cell cycle | 92 | 5.7 | 163 | 14.0 | 120 | 10.3 |
Mitotic cell cycle checkpoint | 23 | 4.1 | 34 | 6.8 | 28 | 6.0 |
Mitotic cell cycle G1/S transition checkpoint | 12 | 2.3 | 14 | 2.6 | 12 | 2.5 |
Mitotic cell cycle G1/S transition DNA damage checkpoint | 11 | 2.4 | 13 | 2.7 | 10 | 2.1 |
Mitotic metaphase/anaphase transition | 11 | 3.5 | 16 | 5.4 | 13 | 4.7 |
Mitotic prometaphase | | | 32 | 9.0 | 23 | 6.7 |
mRNA binding | 19 | 5.4 | 19 | 4.8 | 14 | 3.6 |
Positive regulation of cell cycle | | | 16 | 3.6 | 12 | 2.7 |
Positive regulation of cell cycle process | 12 | 1.2 | 20 | 3.4 | 15 | 2.5 |
Positive regulation of cell proliferation | 59 | 3.4 | 63 | 2.9 | | |
Regulation of cell cycle | 87 | 6.1 | 121 | 9.6 | 93 | 7.5 |
Regulation of cell cycle arrest | 42 | 5.8 | 65 | 10.2 | 51 | 8.4 |
Regulation of cell cycle process | 51 | 4.9 | 86 | 10.5 | 63 | 7.7 |
Regulation of cell proliferation | 105 | 4.1 | 108 | 2.9 | 93 | 3.0 |
Regulation of mitosis | 15 | 3.5 | 21 | 5.2 | 18 | 4.9 |
Regulation of mitotic cell cycle | 34 | 3.1 | 54 | 6.6 | 40 | 4.8 |
Regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle | 14 | 3.2 | 20 | 5.0 | 14 | 3.4 |
RNA binding | 147 | 11.6 | 149 | 10.2 | 143 | 11.8 |
S phase | | | 38 | 7.6 | 28 | 5.7 |
S phase of mitotic cell cycle | 23 | 4.2 | 35 | 7.1 | 26 | 5.4 |
Translation |
Ribonucleoprotein complex | 116 | 13.4 | 126 | 12.9 | 120 | 13.6 |
Ribosome | 69 | 15.2 | 52 | 9.3 | 56 | 11.9 |
Regulation of translation | 25 | 3.7 | | | 26 | 4.2 |
Structural constituent of ribosome | 67 | 16.4 | 47 | 9.3 | 55 | 13.3 |
Translation | 114 | 14.7 | 86 | 8.1 | 103 | 13.2 |
Translation factor activity, nucleic acid binding | 22 | 6.3 | 16 | 3.3 | 19 | 5.3 |
Translation initiation factor activity | 15 | 5.8 | 9 | 2.2 | 13 | 5.0 |
Translational elongation | 54 | 16.4 | 36 | 8.9 | 47 | 14.3 |
Translational initiation | 19 | 6.0 | 12 | 2.4 | 16 | 4.9 |
Translational termination | 50 | 16.2 | 33 | 8.7 | 42 | 13.5 |
Table 3. Growth Factor Downregulation of Cell Differentiation and Lipid Synthesis Ontologies
Table 3. Growth Factor Downregulation of Cell Differentiation and Lipid Synthesis Ontologies
Ontology | EGF Genes ↓ | EGF Z-Score | BPE Genes ↓ | BPE Z-Score | EGF + BPE Genes ↓ | EGF + BPE Z-Score |
Cell differentiation |
Cell differentiation | | | 205 | 2.46 | 194 | 2.58 |
Cornified envelope | 16 | 11.83 | 16 | 11.53 | 16 | 12.01 |
Epidermal cell differentiation | | | 27 | 7.41 | 27 | 7.83 |
Epidermis development | 50 | 8.29 | 47 | 7.16 | 44 | 6.91 |
Epithelial cell differentiation | 41 | 6.3 | 40 | 5.7 | 39 | 5.92 |
Epithelium development | 55 | 4.32 | 55 | 3.94 | 53 | 4.09 |
Keratinization | 13 | 5.68 | 12 | 4.89 | 13 | 5.76 |
Keratinocyte differentiation | 26 | 8.48 | 25 | 7.76 | 25 | 8.18 |
Tissue development | 110 | 4.75 | 108 | 3.95 | 107 | 4.58 |
Lipid synthesis |
Cellular lipid catabolic process | 20 | 3.51 | 18 | 2.61 | 17 | 2.58 |
Cellular lipid metabolic process | 84 | 4.41 | 88 | 4.51 | 84 | 4.6 |
Cholesterol biosynthetic process | | | 12 | 4.68 | 12 | 4.95 |
Cholesterol metabolic process | 15 | 2.27 | 17 | 2.77 | 15 | 2.35 |
Fatty acid β-oxidation | 14 | 5.56 | 12 | 4.28 | 11 | 4.01 |
Fatty acid biosynthetic process | | | 20 | 3.32 | | |
Fatty acid catabolic process | 14 | 4.46 | 12 | 3.31 | 11 | 3.09 |
Fatty acid metabolic process | 38 | 4.05 | 37 | 3.5 | 31 | 2.53 |
Fatty acid oxidation | 18 | 4.94 | 16 | 3.89 | 13 | 2.92 |
Glycerolipid biosynthetic process | 19 | 3.17 | 18 | 2.61 | 18 | 2.92 |
Glycerophospholipid biosynthetic process | 14 | 3.26 | 12 | 2.25 | 12 | 2.49 |
Lipid biosynthetic process | 55 | 3.79 | 56 | 3.59 | 53 | 3.58 |
Lipid catabolic process | 27 | 2.36 | | | | |
Lipid metabolic process | 121 | 5.13 | 124 | 4.91 | 116 | 4.74 |
Lipid modification | 21 | 3.89 | 21 | 3.65 | 18 | 2.96 |
Lipid oxidation | 18 | 4.94 | 16 | 3.89 | 13 | 2.92 |
Phospholipid biosynthetic process | 19 | 3.21 | | | 17 | 2.61 |
Phospholipid metabolic process | 25 | 2.28 | | | | |
These modulatory effects were duplicated in a second series of experiments, which involved the treatment of passage 24 human meibomian gland epithelial cells (n = 3 wells/condition) under proliferating (i.e., EGF + BPE) or differentiating (i.e., serum) conditions for 4 days. The combined growth factors significantly (P < 0.05) increased ontologies associated with cell cycle processes (zsc = 17.1), cell proliferation (zsc = 3.6), DNA replication (zsc = 11.2), ribosome biogenesis (zsc = 8.6), and translation (zsc = 6.9), and reduced those linked to epithelial cell differentiation (zsc = 5.8), tissue development (zsc = 3.0), lipid metabolic processes (zsc = 3.1), and lipid modification (zsc = 4.5).
The influence of growth factors on human meibomian gland epithelial cells also was shown by the analysis of KEGG pathways. As demonstrated in
Table 4, EGF, BPE, and EGF + BPE promoted the activity of cell cycle, DNA replication, and ribosome pathways, and decreased processes related to fatty acid metabolism and peroxisome proliferator–activated receptor (PPAR) signaling (e.g., PPARδ and retinoid X receptor α [RXRα]). Of interest, the growth factors also suppressed pathways associated with acid secretion and lysosomes.
Table 4. Growth Factor Influence on KEGG Pathways in Human Meibomian Gland Epithelial Cells
Table 4. Growth Factor Influence on KEGG Pathways in Human Meibomian Gland Epithelial Cells
Ontology | EGF Genes | EGF Z-Score | BPE Genes | BPE Z-Score | EGF + BPE Genes | EGF + BPE Z-Score |
Upregulation |
Cell cycle | 27 | 4.5 | 38 | 7.1 | 32 | 6.4 |
DNA replication | 11 | 4.3 | 17 | 7.3 | 14 | 6.4 |
Ribosome | 53 | 15.8 | 35 | 8.7 | 43 | 12.9 |
Downregulation |
Collecting duct acid secretion | 6 | 2.4 | 9 | 4.5 | 8 | 3.9 |
Fatty acid metabolism | 11 | 3.8 | 10 | 3.4 | 10 | 3.5 |
Lysosome | 20 | 2.7 | 23 | 3.8 | 18 | 2.4 |
PPAR signaling pathway | 14 | 3.1 | 14 | 3.2 | 12 | 2.5 |
Examples of cell cycle and translation genes upregulated by EGF + BPE treatment of human meibomian gland epithelial cells are listed in
Table 5. An illustration of keratin- and lipid-related genes downregulated by combined growth factor exposure is shown in
Table 6.
Table 5. EGF Plus BPE Upregulation of Selected Cell Cycle- and Translation-Related Genes in Human Meibomian Gland Epithelial Cells
Table 5. EGF Plus BPE Upregulation of Selected Cell Cycle- and Translation-Related Genes in Human Meibomian Gland Epithelial Cells
Accession # | Gene | Ratio | P Value |
Cell cycle |
NM_015714 | G0/G1switch 2 | 16.11 | 0.0206 |
NM_001255 | Cell division cycle 20 homolog | 11.94 | 0.0000 |
NM_001067 | Topoisomerase (DNA) IIα | 10.96 | 0.0028 |
NM_181803 | Ubiquitin-conjugating enzyme E2C | 10.18 | 0.0007 |
NM_004701 | Cyclin B2 | 8.38 | 0.0052 |
NM_005915 | Minichromosome maintenance complex component 6 | 7.37 | 0.0000 |
NM_182776 | Minichromosome maintenance complex component 7 | 6.54 | 0.0001 |
NM_030928 | Chromatin licensing and DNA replication factor 1 | 6.39 | 0.0026 |
NM_002916 | Replication factor C (activator 1) 4 | 5.01 | 0.0012 |
NM_002388 | Minichromosome maintenance complex component 3 | 4.96 | 0.0004 |
NM_001237 | Cyclin A2 | 3.56 | 0.0130 |
NM_004354 | Cyclin G2 | 3.49 | 0.0027 |
NM_002689 | Polymerase (DNA directed), α2 (70kD subunit) | 2.73 | 0.0052 |
NM_001760 | Cyclin D3 | 2.43 | 0.0011 |
NM_053056 | Cyclin D1 | 2.34 | 0.0000 |
NM_199246 | Cyclin G1 | 2.3 | 0.0156 |
NM_057735 | Cyclin E2 | 2.07 | 0.0449 |
NM_182851 | Cyclin B1 interacting protein 1 | 1.99 | 0.0011 |
NM_001759 | Cyclin D2 | 1.43 | 0.0023 |
NM_001259 | Cyclin-dependent kinase 6 | 1.34 | 0.0420 |
NM_000075 | Cyclin-dependent kinase 4 | 1.31 | 0.0401 |
Translation |
NM_138957 | Mitogen-activated protein kinase 1 | 3.61 | 0.0199 |
NM_003246 | Thrombospondin 1 | 3.28 | 0.0006 |
NM_002759 | Eukaryotic translation initiation factor 2-alpha kinase 2 | 2.65 | 0.0154 |
NM_004280 | Eukaryotic translation elongation factor 1 epsilon 1 | 2.16 | 0.0266 |
NM_001402 | Eukaryotic translation elongation factor 1 alpha 1 | 1.81 | 0.0002 |
NM_001568 | Eukaryotic translation initiation factor 3, subunit E | 1.81 | 0.0006 |
Table 6. EGF Plus BPE Downregulation of Selected Keratin- and Lipid-Related Genes in Human Meibomian Gland Epithelial Cells
Table 6. EGF Plus BPE Downregulation of Selected Keratin- and Lipid-Related Genes in Human Meibomian Gland Epithelial Cells
Accession # | Gene | Ratio | P Value |
Keratin |
NM_173086 | Keratin 6C | 251.79 | 0.0000 |
NM_207392 | Keratinocyte differentiation-associated protein | 175.04 | 0.0001 |
NM_005987 | Small proline-rich protein 1A | 166.23 | 0.0000 |
NM_001024209 | Small proline-rich protein 2E | 63.06 | 0.0001 |
NM_003125 | Small proline-rich protein 1B | 62.37 | 0.0000 |
NM_005988 | Small proline-rich protein 2A | 56.75 | 0.0000 |
NM_005557 | Keratin 16 | 52.72 | 0.0000 |
NM_001014291 | Small proline-rich protein 2G | 51.78 | 0.0000 |
NM_182507 | Keratin 80 | 51.01 | 0.0002 |
NM_002964 | S100 calcium binding protein A8 | 10.86 | 0.0001 |
NM_002965 | S100 calcium binding protein A9 | 8.72 | 0.0000 |
Lipid |
NM_004354 | Cyclin G2 | 3.49 | 0.0027 |
NM_001444 | Fatty acid binding protein 5 | 37.75 | 0.0026 |
NM_018404 | ArfGAP with dual PH domains 2 | 25.36 | 0.0001 |
NM_001031615 | Aldehyde dehydrogenase 3 family, member B2 | 21.55 | 0.0001 |
NM_004364 | CCAAT/enhancer binding protein (C/EBP), α | 20.52 | 0.0001 |
NM_004605 | Sulfotransferase family, cytosolic, 2B, member 1 | 17.83 | 0.0001 |
NM_001955 | Endothelin 1 | 10.72 | 0.0002 |
NM_025153 | ATPase, class V, type 10B | 10.02 | 0.0000 |
NM_152443 | Retinol dehydrogenase 12 (all-trans/9-cis/11-cis) | 9.35 | 0.0007 |
NM_002899 | Retinol binding protein 1, cellular | 9.26 | 0.0000 |
To confirm in part the Illumina BeadChip results, selected genes were analyzed by qPCR. This experimental approach verified the regulatory effects of EGF, BPE, and EGF + BPE on laminin, α3; keratin 10; small proline-rich protein 3; and TIMP metallopeptidase inhibitor 1 (
Table 7).
Table 7. Confirmation of Selected BeadChip Gene Expression Results
Table 7. Confirmation of Selected BeadChip Gene Expression Results
Gene | EGF | EGF | BPE | BPE | EGF + BPE | EGF + BPE |
Array | qPCR | Array | qPCR | Array | qPCR |
Upregulation |
LAMA3 | 11.2 | 8.2 | 9.6 | 5.7 | 10.3 | 4.9 |
TIMP1 | 11.7 | 13.6 | 4.8 | 4.9 | 8.4 | 6.9 |
Downregulation |
KRT10 | 10.6 | 10.8 | 10.2 | 13.7 | 12.4 | 16.3 |
SPRR3 | 14.0 | 10.5 | 1.6 | 3.0 | 22.9 | 11.4 |
The influence of EGF and BPE on human meibomian gland epithelial cells was not completely the same. In fact, BPE significantly (P < 0.05) increased the expression of 1088 genes compared to EGF. In turn, EGF significantly (P < 0.05) upregulated the activity of 918 genes relative to BPE. Major differences occurred between these growth factors in the extent of their impact on ontologies and KEGG pathways. For example, BPE induced far greater effects on cell cycle processes (zsc = 14.1) and DNA replication (zsc = 8.5), whereas EGF exerted a stronger influence on ribosomes (zsc = 11.3) and translation (zsc = 11.9).
Impact of EGF + BPE on Lipid Accumulation in Human Meibomian Gland Epithelial Cells
To test our hypothesis that growth factors do not promote differentiated cell functions, such as lipid accumulation, we induced human meibomian gland epithelial cells to proliferate with EGF + BPE, or to differentiate with serum. In addition, to demonstrate the specificity of the lipid-related response of meibomian (i.e., sebaceous-like) cells, we exposed human conjunctival epithelial cells to the same conditions. Following 7 days of treatment, cells were fixed and stained with LipidTox (green) and DAPI (red).
As shown in
Figure 4, EGF + BPE exposure had no apparent effect on cellular lipid levels compared to those of vehicle-treated cells. In contrast, serum treatment elicited a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated in human conjunctival epithelial cells (
Fig. 4).
Our studies showed that EGF and BPE, alone or in combination, stimulated a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects were associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and PPAR signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. Overall, our results supported our hypothesis that growth factors stimulate human meibomian gland epithelial cells to proliferate through processes involving gene regulation.
Our finding that EGF and BPE increased the proliferation, but not the differentiation, of human meibomian gland epithelial cells is consistent with the responses of other cell types to these growth factors.
2–11 EGF + BPE induced the highest rate of proliferation, followed by BPE alone, then EGF alone. In contrast, MCDB and serum-containing media supported minimal to no proliferation. SFM permitted slight growth in late-, but not early-passage cells. Of particular interest, the rapidity and magnitude of the proliferative response to EGF and BPE was influenced by the cell passage number. Late-passage (i.e., 50) human meibomian gland epithelial cells proliferated more quickly than early-passage (i.e., 16) cells, despite being exposed to the same culture conditions. This difference may reflect a positive selection of faster-growing cells,
17,18 as well as the development of an altered karyotype,
12 with increasing passage number. We found previously that these human meibomian gland epithelial cells express slight changes in karyotype after passage 34.
12 For this reason, we performed our microarray analyses on early-passage cells, which feature a normal karyotype.
12
The growth factor stimulation of human meibomian gland epithelial cell proliferation was associated with a significant upregulation of genes linked to DNA replication, cell cycle, ribosomes, and translation. Stimulated genes related to DNA replication included DNA directed polymerase, minichromosome maintenance complex components, replication factor C, and replication proteins. EGF and BPE also increased the expression of genes for cyclin B2 and D1-3, cyclin-dependent kinase (CDK) 4 and 6, and E2F transcription factor, and downregulated those genes encoding CDK inhibitors. Collectively, these genes are the major molecular components that drive cell cycle progression. Cyclin is a regulatory subunit that modulates the activation state of the catalytic subunit CDK in the cyclin-CDK complex.
19 Cyclin B is involved in the control of G2/M transition and mitosis.
19 Cyclin Ds are proto-oncogenic components of the retinoblastoma pathway, and are able to form complexes with Cdk2, Cdk4, Cdk5, and Cdk6.
20 Indeed, the accumulation of cyclin D1-Cdk4/6 complexes is of great importance for cell cycle progression.
21 These complexes promote release of E2F transcription factors, which allow the transcription of genes required for the transition from G1 through the S phase.
22–24 The growth factor upregulation of ribosome- and translation-associated genes was to be expected, given that ribosome biogenesis consumes approximately 80% of the energy of a proliferating cell,
25,26 and that ribosomal protein mRNAs monopolize the translational capacity of cells.
26
We discovered that EGF and BPE cause a significant decrease in the expression of genes related to cell differentiation and keratinization, such as small proline-rich proteins (SPRR), keratins, the S100 calcium binding proteins A8 (S100A8) and A9 (S100A9), and keratinocyte differentiation-associated protein. The SPRR are precursors of the cornified cell envelope of terminally differentiating, stratified squamous epithelia and influence the rigidity of this envelope.
27–31 The SPRR, whether alone
27 or combined with S100A8 and A9,
32 are known to promote keratinization in the conjunctiva
33,34 and elsewhere in the body.
35,36 Of particular interest, the expression of SPRR, S100A8, and S100A9 genes is increased significantly in meibomian glands of patients with meibomian gland dysfunction (MGD).
13 It is possible application of these growth factors might be able to suppress the keratinization process, which is a primary cause of MGD.
2,37
We also found that EGF and BPE significantly reduce the expression of genes associated with lipid biosynthesis and PPAR signaling. This growth factor-induced gene effect could account for the profound lack of lipid accumulation in proliferating meibomian (this study)
12 and sebaceous
38 gland epithelial cells. As concerns PPARs, these ligand-activated nuclear factors form heterodimers with retinoid X receptors (RXR) to regulate transcription.
39–42 PPARs have been identified in mitochondria, peroxisomes, and microsomes of sebocytes, and are important modulators of multiple lipid metabolic genes.
43 There are three subclasses of PPARs: α, β/δ, and γ, each with different tissue localizations and transcriptional activities.
39,44,45 PPARδ is a potent metabolic regulator in various tissues, such as fat, skeletal muscle, and the heart. PPARδ enhances fatty acid catabolism and energy uncoupling in adipose tissue, and suppresses macrophage-derived inflammation.
46,47 PPARδ also regulates the late stages of sebocyte differentiation and stimulates sebaceous lipid synthesis.
48 In our study, we demonstrated that EGF and BPE significantly downregulate the genes encoding PPARδ and RXRα in human meibomian gland epithelial cells. These growth factor actions possibly may contribute to the suppression of lipid accumulation in these cells.
The influence of EGF and BPE on human meibomian gland epithelial cells was not identical. There were significant differences between these factors in terms of their impact on specific genes, ontologies, and KEGG pathways. For example, BPE induced far greater effects on genes related to cell cycle processes, compared to EGF. This BPE influence may reflect the fact that pituitary extracts should contain adrenocorticotrophic hormone, fibroblast growth factor, growth hormone, insulin-like growth factor 1, thyroid-stimulating hormone, α-melanocyte stimulating hormone, and transforming growth factor-α,
49,50 as well as EGF, all of which have been shown to stimulate the proliferation of sebocytes.
37 However, this stimulatory effect of BPE may be countered somewhat by β-endorphin, which also is present in the pituitary and reportedly decreases sebocyte proliferation.
51
Our results demonstrated that serum-induced differentiation, but not growth factor-elicited proliferation, promotes a striking accumulation of lipids within human meibomian gland epithelial cells. This response, which was not found in human conjunctival epithelial cells, also may occur after a decrease in the proliferation of meibomian gland epithelial cells
12 and sebocytes.
38 These findings are consistent with earlier observations that neutral lipids accumulate in sebaceous gland epithelial cells during differentiation.
38,52
Overall, our investigation demonstrated that EGF and BPE exert a significant effect on the proliferation and gene expression of human meibomian gland epithelial cells.
The authors thank Stephen M. Richards (Boston, MA) for his technical assistance, and Ilene K. Gipson and Sandra Michaud (Boston, MA) for the human conjunctival epithelial cells.
Supported by grants from NIH (EY05612) and Alcon Research, Ltd.
Disclosure: S. Liu, None; W.R. Kam, None; J. Ding, None; M.P. Hatton, None; D.A. Sullivan, Alcon Research, Ltd. (F)