Yonsei Med J.  2017 Mar;58(2):423-431. 10.3349/ymj.2017.58.2.423.

Role of TGFBIp in Wound Healing and Mucin Expression in Corneal Epithelial Cells

• Maeng YS
• Lee GH
• Lee B
• Choi SI
• Kim TI
• Kim EK

• KMID: 2427133
• DOI: http://doi.org/10.3349/ymj.2017.58.2.423

Abstract

PURPOSE
Transforming growth factor-β-induced protein (TGFBIp) is highly expressed in the cornea, and mutant TGFBIp induces corneal diseases. However, the function of TGFBIp in cornea epithelium is not fully investigated. Here, we tested the importance of TGFBIp in regulation of gene expression and corneal epithelial cell (CEC) activity.
MATERIALS AND METHODS
The effect of TGFBIp on CEC activity was analyzed by cell migration, adhesion, proliferation and wound healing assay. Analysis of gene expression was examined by western blot and quantitative reverse transcription PCR.
RESULTS
The results demonstrated that TGFBIp increased adhesion, migration, proliferation, and wound healing of CECs. Analysis of gene expression presented that TGFBIp-stimulated CECs exhibited increased expression of mucin family genes, such as MUC1, -4, -5AC, and -16. Furthermore, TGFBIp treatment increased the expression of MUC1, -4, -5AC, -7, and -16 in conjunctival epithelial cells. TGFBIp also increased the activity of intracellular signaling molecules ERK and AKT in CECs. Using pharmacologic inhibitors of ERK and AKT, we showed that the expression of mucin genes by TGFBIp is mediated by the activation of ERK and AKT signaling.
CONCLUSION
Our findings demonstrate that the locally generated TGFBIp in the cornea may contribute to wound healing of CECs by enhancing the migration, adhesion, and proliferation of CECs. In addition, our results suggest that TGFBIp has a protective effect on ocular surfaces by inducing the expression of mucin genes in corneal and conjunctival epithelial cells. These data suggest that TGFBIp is a useful therapeutic target for patients with corneal wounds.

Keyword

TGFBIp; mucin; cornea; epithelial cells

MeSH Terms

Blotting, Western
Cell Adhesion
Cell Movement/drug effects
Cell Proliferation
Cells, Cultured
Conjunctiva/cytology
Cornea/*cytology
Epithelial Cells/*metabolism
Extracellular Matrix Proteins/*physiology
Gene Expression
Humans
Mucins/genetics/*metabolism
Signal Transduction
Transforming Growth Factor beta/*physiology
Wound Healing/*physiology
Extracellular Matrix Proteins
Mucins
Transforming Growth Factor beta

Figure

• 

  Fig. 1 TGFBIp increases the migration, adhesion, proliferation, and wound healing of SV40-CECs. (A and B) Effect of TGFBIp on SV40-CECs migration. SV40-CECs were treated with TGFBIp or fibronectin and migration was quantified. (C and D) Effect of TGFBIp on SV40-CECs adhesion. SV40-CECs were treated with TGFBIp and cell adhesion was quantified. (E) Effect of TGFBIp on SV40-CECs proliferation. SV40-CECs were incubated with TGFBIp (2.5–10 µg/mL) for 72 hours and cell proliferation was assessed via MTT assay. (F and G) Effect of TGFBIp on SV40-CECs wound healing. SV40-CECs were scratched with micropipette tips and treated with TGFBIp. Images were captured at 0 and 16 hours after wounding. All data are presented as mean±SE from three independent experiments performed in duplicates or quintuplicate. **p<0.01 vs. control or fibronectin. TGFBIp, transforming growth factor-β-induced protein; CECs, corneal epithelial cells; SE, standard error; Cont, control.

• 

  Fig. 2 TGFBIp increases the migration, adhesion, proliferation, wound healing of primary CECs. (A) Effect of TGFBIp on primary CECs migration. Primary CECs were treated with TGFBIp and migration was quantified. (B) Effect of TGFBIp on primary CECs adhesion. Primary CECs were treated with TGFBIp and cell adhesion was quantified. (C) Effect of TGFBIp on primary CECs proliferation. Primary CECs were incubated with TGFBIp (2.5–10 µg/mL) for 72 hours and cell proliferation was assessed via MTT assay. (D) Effect of TGFBIp on primary CECs wound healing. Primary CECs were scratched with micropipette tips and treated with TGFBIp. For quantitative analysis, distances between front lines were measured using the ImageJ software. All data are presented as mean±SE from three independent experiments performed in duplicates or quintuplicate. **p<0.01 vs. control. TGFBIp, transforming growth factor-β-induced protein; CECs, corneal epithelial cells; SE, standard error; Cont, control.

• 

  Fig. 3 TGFBIp increases the expression of mucins in SV40-CECs. (A) The temporal expression of each gene in SV40-CECs treated with TGFBIp was determined by qRT-PCR. (B) The mRNA level of each gene in SV40-CECs without TGFBIp treatment was determined by qRT-PCR. All results were normalized to GAPDH. **p<0.01 vs. control. TGFBIp, transforming growth factor-β-induced protein; CECs, corneal epithelial cells; qRT-PCR, quantitative reverse transcription PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 4 TGFBIp treatment induces the expression of mucins in primary CECs. (A) The temporal expression of each gene in primary CECs treated with TGFBIp was determined by qRT-PCR. (B) The mRNA level of each gene in primary CECs without TGFBIp treatment was determined by qRT-PCR. All results were normalized to those for GAPDH. **p<0.01 vs. control. TGFBIp, transforming growth factor-β-induced protein; CECs, corneal epithelial cells; qRT-PCR, quantitative reverse transcription PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 5 TGFBIp regulates the expression of mucins through the ERK and AKT signaling pathways in CECs. (A) SV40-CECs were treated with TGFBIp (10 µg/mL) for indicated period of time and cell lysates were subjected to Western blot analysis. (B) The relative ratios were normalized by arbitrarily setting the phosphorylation ratio at time 0 as 1. Similar results were obtained from three additional experiments (**p<0.01 vs. time 0). (C) SV40-CECs were pre-incubated with or without PD98059 (10 µM) or Wortmannin (100 nM), then stimulated with TGFBIp (10 µg/mL) for 6 hours. The mRNA level of each gene in CECs was determined by qRT-PCR. All results were normalized to GAPDH. **p<0.01 vs. control or TGFBIp. (D) SV40-CECs were pre-incubated with or without PD98059 (10 µM) or Wortmannin (100 nM) and then stimulated with TGFBIp (10 µg/mL) for 10 minutes. Cell lysates were subjected to Western blot. (E and F) SV40-CECs were pre-incubated with or without PD98059 (10 µM) or Wortmannin (100 nM) and then stimulated with TGFBIp (10 µg/mL) for 8 hours. Protein levels of mucin 1 and mucin 4 were analyzed by Western blot analysis. The relative ratios were normalized by arbitrarily setting the expression ratio for the control as 1. Similar results were obtained from three additional experiments (**p<0.01 vs. TGFBIp). TGFBIp, transforming growth factor-β-induced protein; CECs, corneal epithelial cells; qRT-PCR, quantitative reverse transcription PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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