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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Exp Eye Res. 2016 Mar 16;146:233–241. doi: 10.1016/j.exer.2016.03.011

Molecular Insights on the Effect of TGF-β1/-β3 in Human Corneal Fibroblasts

Xiaoqing Guo a, Audrey E K Hutcheon a, James D Zieske a
PMCID: PMC4893894  NIHMSID: NIHMS774355  PMID: 26992778

Abstract

Transforming growth factor β (TGF-β) plays a critical role in wound healing and the pathogenesis of fibrosis (scarring). Three isoforms of TGF-β have been identified in mammals. Previous studies have shown that the addition of TGF-β1 (T1) or -β2 (T2) to human corneal fibroblasts (HCF) cultured in a 3-dimensional construct resulted in a fibrotic matrix, while the addition of TGF-β3 (T3) resulted in the production of enhanced non-fibrotic matrix as compared to control (Vitamin C [VitC] only). In the current investigation, we undertook the molecular comparison of fibrosis-related gene expression in T1 or T3-treated HCF to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. HCF were cultured in 100mm dishes in basic medium (Eagles minimum essential medium [EMEM] with 10% fetal bovine serum [FBS]). At 70-80% confluency, cells were exposed to basic medium with 0.5mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (VitC) ± 2ng/ml of T1 or T3. After 4 hours or 3 days, cells were harvested, and mRNA or protein was isolated. Fibrosis related mRNA levels were assayed using a commercial qRT-PCR Array. Selected proteins were examined using Western blotting (WB). Experiments were performed 6 times for the qRT-PCR and 4 times for WB for each condition. qRT-PCR results showed that most of the fibrosis-related genes were up or downregulated in HCF exposed to T1 or T3 as compared with VitC control. At 4 hours, only Smad7 expression was significantly altered in T3-treated HCF, compared to T1, and at 3 days, five genes were altered. WB confirmed that T1 significantly decreased Smad7 expression compared to T3 and control, and that the expression of thrombospondin-1 in T3-stimulated HCF was enhanced compared to T1-treated cells. Finally, both T1 and T3 decreased Smad3 expression dramatically at both time points. At early time points, T1 and T3 have similar effects on expression of fibrosis related genes; however, with a longer exposure, an increasing number of genes were differentially expressed. Interestingly, most of the differentially expressed gene products are secreted by the cells and may be related to the modulation of extracellular matrix.

Keywords: TGF-beta1, TGF-beta3, human corneal fibroblasts, gene array, Smad7, Smad3, Thrombospondin-1, fibrosis

1. Introduction

The cornea is a transparent tissue covering the front of the eye. It not only serves as a barrier against dirt and germs from the environment, but also plays a key role in vision. There are three main layers in the cornea: 1) epithelium, the most superficial layer; 2) stroma, the middle and thickest layer populated with quiescent keratocytes; and 3) endothelium, a single layer of cells located between the stroma and the aqueous humor (Wilson et al., 2012). In the cornea, quiescent stromal keratocytes play a critical role in keeping the cornea clear and refractive (Nishida, 1995). Injury or wounds to the cornea, especially to the stroma, frequently lead to corneal keratocyte activation, migration, and differentiation into fibroblasts and myofibroblasts, which then produce corneal haze or fibrosis (scarring). These complications of wound healing are the major causes of decreased visual quality and vision loss worldwide (Jester, 2008; Jester et al., 1999; Wilson et al., 2012).

Corneal wound healing is regulated by various growth factors, including, but not exclusive to, epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor β (TGF-β), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), the interleukins (IL-1, IL-6, IL-8), tumor necrosis factor alpha (TNF-α), and secreted protein, acidic and rich in cystine (SPARC) (Imanishi et al., 2000; Schultz et al., 1992; Wilson et al., 2012; Yu et al., 2010). Evidence has accumulated indicating that TGF-β is one of the key regulators of fibrosis in the cornea, as well as in other tissues (Branton and Kopp, 1999; Cutroneo, 2007; Karamichos et al., 2010; Leask and Abraham, 2004; Park et al., 2001; Ruiz-Ortega et al., 2007; Tatler and Jenkins, 2012; Wilson et al., 2012). Three closely related isoforms of TGF-β—TGF-β1, -β2, and -β3 (T1, T2, and T3, respectively)—have been identified in mammals and are encoded by three distinct genes with 64-85% amino acid sequence homology (Schiller et al., 2004; Wilson et al., 2012). All three isoforms are synthesized as precursor molecules containing a propeptide region and the TGF-β homodimer. During processing, they are cleaved to form an active 25kDa growth factor and a latency-associated peptide (LAP). Although cleaved, these two pieces remain a small complex attached by noncovalent bonds. This small complex then binds another protein, latent TGF-β-binding protein (LTBP), to form a large complex before it is secreted. After its secretion, it remains in the extracellular matrix (ECM) as an inactive complex containing the LAP and LTBP, which prevents the complex from binding to its receptors. The inactive complex of TGF-β is further processed through proteases, acid, reactive oxygen species (ROS), thrombospndin-1 (THBS1), and αV containing integrins in order to release active TGF-β (Annes et al., 2004; Carrington et al., 2006; Daniel et al., 2007; Shi et al., 2011; Taylor, 2009). Since different cellular mechanisms require distinct levels of TGF-β signaling, the inactive complex of this growth factor gives opportunity for a proper mediation of TGF-β signaling (Annes et al., 2003).

All three TGF-β isoforms signal through the same cell surface receptors and have similar cellular targets (Letterio and Roberts, 1998). Signaling occurs once the activated TGF-β is directly bound to its receptor (TGF-βRII or TRII), which then forms a complex with the type I receptor (TRI). The canonical signaling pathway for TGF-β is the Smad-cascade. Three functional classes of Smads are involved in this pathway: the receptor-activated Smad (R-Smads: Smad1, 2, 3, 5, and 8), the common mediator Smad (Co-Smad: Smad4), and the inhibitory Smads (I-Smads: Smad6 and 7) (Euler-Taimor and Heger, 2006; Massague et al., 2005). T1 and T3 exhibit a substantial overlap in biological functions, such as inhibition of proliferation of different cell types, regulation of immune response, and stimulation of ECM formation (Olsson et al., 2000). Among them, T1 is the most commonly documented isoform. It has been found to promote the transition of quiescent corneal keratocytes to activated fibroblasts to myofibroblasts in a rabbit corneal wound (Jester et al., 2002), and induce fibrosis and scar formation after wounding (Ask et al., 2008; Shah et al., 1995); while T3 has been shown in skin, lung, and kidney models to be anti-fibrotic, and in some cases, to stimulate scar-free healing (Ask et al., 2008; Carrington et al., 2006; Eslami et al., 2009; Hosokawa et al., 2003; Occleston et al., 2008; Ohno et al., 2012; Saika, 2006; So et al., 2011; Waddington et al., 2010). We have developed a cell-based 3-dimensional (3D) corneal stromal construct with a self-assembled matrix that mimics the in vivo stromal development (Karamichos et al., 2011). Interestingly, addition of T1 or T2 to the culture resulted in a fibrotic matrix; however, addition of T3 resulted in enhanced matrix production compared to control, with no evidence of fibrotic markers, such as collagen type III and α-smooth muscle actin (SMA, also known as ACTA2). It remains unclear why T1 and T3 stimulate different responses. Insights of molecular mechanisms of T1 and T3 and their relationship with ECM deposition by HCF would provide effective options for understanding the potential anti-scarring effects of T3. In the current study, we compared the fibrosis-related mRNA and protein level changes in HCFs treated with T1 or T3.

2. Material and Methods

This study adheres to the Declaration of Helsinki and was approved by the institutional review board for human subjects at the Schepens Eye Research Institute/Massachusetts Eye and Ear.

2.1. Cell Culture

The isolation and culture of human corneal fibroblasts (HCF) was described previously (Guo et al., 2007b). Briefly, human corneal stromal explants from eyes received from National Disease Research Interchange (NDRI; Philadelphia, PA) were put in 6-well plates in basic medium (Eagles minimum essential medium with 10% FBS) and incubated at 37°C with 5% CO2 until sufficient HCF migrated from the explants. The HCF then were seeded in 100mm dishes in basic medium until they reached 70-80% confluency, at which time the cells were exposed to Vitamin C (VitC) medium (basic medium plus 0.5mM 2-O-α-D-glucopyranosyl-L-ascorbic acid) ± 2ng/ml of T1 or T3, and harvested after 4 hours or 3 days. This concentration of TGF-β has been previously used by us and shown to activate TGF-β receptor (Zieske et al., 2001). Experiments were performed 6 times for quantitative real-time polymerase chain reaction (qRT-PCR) and 4 times for western blot for each condition.

2.2. Indirect Immunofluorescence

HCF (1×105) were plated on 4-well chamber slides, and grown for 3 days in VitC medium ± T1 or T3. The medium was removed and cells were fixed with 100% Methanol at -20°C for 10 minutes. Indirect-immunofluorescence (IF) was performed as previously described (Zieske et al., 1994). Cells were incubated with primary antibody against SMA (Dako North America, Inc.; Carpinteria, CA) and then the corresponding secondary antibody (donkey anti-mouse IgG-FITC: Jackson ImmunoResearch; West Grove, PA). After which, the cells were coverslipped with Vectashield mounting media containing DAPI (Vector Laboratory; Burlingame, CA), a nuclear counterstain. Cells were then examined with a Nikon Eclipse E800 equipped with an Andor Clara E camera and Nikon NIS Elements for Basic Research (Micro Video Instruments, Inc.; Avon, MA) and photographed. ImageJ (v. 1.45s; National Institutes of Health; Bethesda, MD: http://imagej.nih.gov/ij) was used to count the number of SMA-positive cells and nuclei stained with DAPI. The experiment was performed 4 times and for each time, at least 5 areas in each well were counted and averaged.

2.3. RNA Isolation and qRT-PCR

qRT-PCR experiments were performed to screen for fibrosis-related genes that may be differentially regulated in HCF stimulated with either T1 or T3. Total RNA was isolated from HCF using Trizol (Invitrogen; Carlsbad, CA) according to manufacturer's protocol, and further total RNA purification was performed using RNeasy Mini kit (Qiagen; Valencia, CA). Total RNA (1μg) then was reverse transcribed to cDNA using the RT2 first-strand kit (Qiagen), and qRT-PCR was performed according to manufacturer's instructions for the Human Fibrosis RT2 PCR Array (Qiagen). The Array was run on an Eppendorf Multiplex 2 Real Time PCR machine (Eppendorf; Hauppauge, NY), and the data was analyzed with the SABiosciences RT2 Profiler PCR Array Data Analysis Software, v.3.4.

2.4. SDS-PAGE and Western Blot Analysis

Protein isolation and western blot analysis were performed as previously described (Zieske et al., 2001). In brief, protein from HCF treated ± T1 or T3 in VitC medium was extracted with RIPA buffer (10mM Tris, 150nM NaCl, 1% deoxycholic acid, 1% Triton X, 0.1% SDS, 1mM EDTA) plus protease inhibitors (aprotinin, PMSF, and sodium orthovanadate). Protein concentration was determined using a protein assay kit (Bio-Rad Protein Assay; Hercules, CA), and equal amount of protein (15-30μg/lane) from each sample was loaded onto 4-20% gradient Tris-Glycine Gels (Invitrogen). Proteins were transferred onto PVDF membranes (Invitrogen), and the transfer was confirmed by staining the membrane with 0.1% Ponceau S solution (Sigma-Aldrich; St. Louis, MO). Membranes then were incubated with primary antibodies against either type I collagen (abcam; Cambridge, MA), Smad3 (Zymed; Grand Island, NY), Smad7 (LifeSpan BioSciences, Inc.; Seattle, WA), or thrombospondin-1 (THBS1: Thermo Scientific; Waltham, MA). Protein bands were detected by Chemiluminescence (Milipore; Billerica, MA) after exposure to film. Band intensities were quantified with imaging software (ImageJ, v.1.45s: National Institutes of Health, USA; http://imagej.nih.gov/ij).

2.5. Statistical Analysis

All experiments were repeated 4-6 times and data was analyzed for significance (p<0.05 to p<0.001) using the Student's t-test and Dunnett's Multiple Comparison test (GraphPad Prism v.5.0b; La Jolla, CA).

3. Results

3.1. Indirect-Immunofluorescence

We have previously observed that the addition of T1 to 4-week HCF cultures stimulated an increase of SMA-positive cells that was far greater than seen upon T3 stimulation (Karamichos et al., 2011). To confirm that this effect was also seen in short term cultures, T1 or T3 were added to HCF and the number of SMA-positive cells were observed by indirect-immunofluorescence and quantified (Figure 1). Figure 1 shows that the number of SMA-positive cells was significantly increased (p<0.05) by both T1 (Fig. 1B and D) and T3 (Fig. 1C and D) as compared with control (Fig. 1A and D), with T1 having a higher number of SMA-positive cells than T3.

Figure 1. Indirect-immunofluorescent analysis of α-smooth muscle actin (SMA) in human corneal fibroblasts (HCF) ± TGF-β1 (T1) or TGF-β3 (T3).

Figure 1

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Representative indirect-imuunofluorescent images show that in Control (VitC) samples (A), there are only a few SMA-positive cells present; however, with the addition of T1 (B), the number of SMA-positive cells increased significantly (D: *p<0.05) as compared with control (A and D). T3-stimulated cells (C) also showed a significant increase of SMA-positive cells (D: *p<0.05) as compared with control (A and D), however, to a lesser extent than T1 (B and D).

3.2. qRT-PCR

Since T1 and T3 appeared to have differing effects on the increase in the number of SMA-positive cells, we used a commercial PCR array to examine genes associated with fibrosis (Table 1). The Human Fibrosis PCR Array contained 84 key genes involved in the dysregulated tissue remodeling during repair and healing of wounds. Numerous statistically significant changes were seen in all conditions examined. As seen in Table 2, in response to T1 compared with Control, 25 genes were upregulated and 4 downregulated by 4 hours, while 10 were upregulated and 12 downregulated by 3 days. In response to T3, 16 genes were upregulated and 7 were downregulated by 4 hours, and 19 were upregulated and 8 were downregulated by 3 days. Several patterns of gene regulation were observed. One of the most dramatic was INHBE (inhibin, beta E), whose mRNA in T1-treated samples changed dramatically from a decrease of about 3 fold at 4 hours to an increase of 127 fold at 3 days. Other genes, such as Smad 7, ITGB6 (Integrin, beta 6), CTGF (connective tissue growth factor), PDGFA (platelet-derived growth factor alpha), and SERPINE 1 (serpin peptidase inhibitor, clade E [nexin, plasminogen activator inhibitor type 1], member 1), maintained a significant upregulation in both 4-hour and 3-day T1-stimulated samples. Still others went from a significant increase at 4 hours to a significant decrease by 3 days with T1 stimulation, these genes include, ITGB3 (Integrin, beta 3), MMP1 (matrix metalloproteinase 1), PLAU (Plasminogen Activator, Urokinase), CCL2 (Chemokine [C-C motif] ligand 2) and CCL11. In T3-treated samples, COL3A1 (collagen type III, alpha 1) mRNA changed from -2 fold at 4 hours to +4 fold at 3 days, whereas, ITGB6, ITGAV (integrin, alpha V), EDN1 (endothelin 1), CTGF, VEGFA (vascular endothelial growth factor A), PDGFA, SERPINE1, THBS1 all remained upregulated in 4-hour and 3-day samples. Only CCL11 mRNA decreased with time, changing from 6.3 fold at 4 hours to -8.7 fold by 3 days.

Table 1. Genes on the Human Fibrosis RT2 PCR Array (Qiagen).

Symbol Description Gname
ACTA2 Actin, alpha 2, smooth muscle, aorta AAT6/ACTSA
AGT Angiotensinogen (serpin peptidase inhibitor, clade A, member 8) ANHU/FLJ92595/FLJ97926/SERPINA8
AKT1 V-akt murine thymoma viral oncogene homolog 1 AKT/MGC99656/PKB/PKB-ALPHA/PRKBA/RAC/RAC-ALPHA
BCL2 B-cell CLL/lymphoma 2 Bcl-2
BMP7 Bone morphogenetic protein 7 OP-1
CAV1 Caveolin 1, caveolae protein, 22kDa BSCL3/CGL3/MSTP085/VIP21
CCL11 Chemokine (C-C motif) ligand 11 MGC22554/SCYA11
CCL2 Chemokine (C-C motif) ligand 2 GDCF-2/HC11/HSMCR30/MCAF/MCP-1/MCP1/MGC9434/SCYA2/SMC-CF
CCL3 Chemokine (C-C motif) ligand 3 G0S19-1/LD78ALPHA/MIP-1-alpha/MIP1A/SCYA3
CCR2 Chemokine (C-C motif) receptor 2 CC-CKR-2/CCR2A/CCR2B/CD192/CKR2/CKR2A/CKR2B/CMKB R2/FLJ78302/MCP-1-R/MGC103828/MGC111760/MGC168006
CEBPB CCAAT/enhancer binding protein (C/EBP), beta C/EBP-beta/CRP2/IL6DBP/LAP/MGC32080/NF-IL6/TCF5
COL1A2 Collagen, type I, alpha 2 OI4
COL3A1 Collagen, type III, alpha 1 EDS4A/FLJ34534
CTGF Connective tissue growth factor CCN2/HCS24/IGFBP8/MGC102839/NOV2
CXCR4 Chemokine (C-X-C motif) receptor 4 CD184/D2S201E/FB22/HM89/HSY3RR/LAP3/LCR1/LE STR/NPY3R/NPYR/NPYRL/NPYY3R/WHIM
DCN Decorin CSCD/DSPG2/PG40/PGII/PGS2/SLRR1B
EDN1 Endothelin 1 ET1/HDLCQ7/PPET1
EGF Epidermal growth factor HOMG4/URG
ENG Endoglin CD105/END/FLJ41744/HHT1/ORW/ORW1
FASLG Fas ligand (TNF superfamily, member 6) APT1LG1/CD178/CD95-L/CD95L/FASL/TNFSF6
GREM1 Gremlin 1 CKTSF1B1/DAND2/DRM/GREMLIN/IHG-2/MGC126660
HGF Hepatocyte growth factor (hepapoietin A; scatter factor) DFNB39/F-TCF/HGFB/HPTA/SF
IFNG Interferon, gamma IFG/IFI
IL10 Interleukin 10 CSIF/IL-10/IL10A/MGC126450/MGC126451/TGIF
IL13 Interleukin 13 ALRH/BHR1/IL-13/MGC116786/MGC116788/MGC116789/P600
IL13RA2 Interleukin 13 receptor, alpha 2 CD213A2/CT19/IL-13R/IL13BP
IL1A Interleukin 1, alpha IL-1A/IL1/IL1-ALPHA/IL1F1
IL1B Interleukin 1, beta IL-1/IL1-BETA/IL1F2
IL4 Interleukin 4 BCGF-1/BCGF1/BSF-1/BSF1/IL-4/MGC79402
IL5 Interleukin 5 (colony-stimulating factor, eosinophil) EDF/IL-5/TRF
ILK Integrin-linked kinase DKFZp686F1765/ILK-2/P59
INHBE inhibin, beta E MGC4638
ITGA1 Integrin, alpha 1 CD49a/VLA1
ITGA2 Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) BR/CD49B/GPIa/VLA-2/VLAA2
ITGA3 Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor) CD49C/FLJ34631/FLJ34704/GAP-B3/GAPB3/MSK18/VCA-2/VL3A/VLA3a
ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) CD51/DKFZp686A08142/MSK8/VNRA
ITGB1 Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) CD29/FNRB/GPIIA/MDF2/MSK12/VLA-BETA/VLAB
ITGB3 Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) CD61/GP3A/GPIIIa
ITGB5 Integrin, beta 5 FLJ26658
ITGB6 Integrin, beta 6 -
ITGB8 Integrin, beta 8 -
JUN Jun proto-oncogene AP-1/AP1/c-Jun
LOX Lysyl oxidase MGC105112
LTBP1 Latent transforming growth factor beta binding protein 1 MGC163161
MMP1 Matrix metallopeptidase 1 (interstitial collagenase) CLG/CLGN
MMP13 Matrix metallopeptidase 13 (collagenase 3) CLG3/MANDP1
MMP14 Matrix metallopeptidase 14 (membrane-inserted) 1/MMP-14/MMP-X1/MT-MMP/MT-MMP 1/MT1-MMP/MT1MMP/MTMMP1
MMP2 Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) CLG4/CLG4A/MMP-II/MONA/TBE-1
MMP3 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) CHDS6/MGC126102/MGC126103/MGC126104/MMP-3/SL-1/STMY/STMY1/STR1
MMP8 Matrix metallopeptidase 8 (neutrophil collagenase) CLG1/HNC/MMP-8/PMNL-CL
MMP9 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) CLG4B/GELB/MANDP2/MMP-9
MYC V-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39/c-Myc
NFKB1 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 DKFZp686C01211/EBP-1/KBF1/MGC54151/NF-kappa-B/NF-kappaB/NFKB-p105/NFKB-p50/NFkappaB/p105/p50
PDGFA Platelet-derived growth factor alpha polypeptide PDGF-A/PDGF1
PDGFB Platelet-derived growth factor beta polypeptide FLJ12858/PDGF2/SIS/SSV/c-sis
PLAT Plasminogen activator, tissue DKFZp686I03148/T-PA/TPA
PLAU Plasminogen activator, urokinase ATF/UPA/URK/u-PA
PLG Plasminogen DKFZp779M0222
SERPINA1 Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 A1A/A1AT/AAT/MGC23330/MGC9222/PI/PI1/PRO2275/ alpha1AT
SERPINE1 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 PAI/PAI-1/PAI1/PLANH1
SERPINH1 Serpin peptidase inhibitor, clade H (heat shock protein 47), member 1, (collagen binding protein 1) AsTP3/CBP1/CBP2/HSP47/PPROM/RA-A47/SERPINH2/gp46
SMAD2 SMAD family member 2 JV18/JV18-1/MADH2/MADR2/MGC22139/MGC34440/hMAD-2/hSMAD2
SMAD3 SMAD family member 3 DKFZp586N0721/DKFZp686J10186/HSPC193/HsT174 36/JV15-2/MADH3/MGC60396
SMAD4 SMAD family member 4 DPC4/JIP/MADH4
SMAD6 SMAD family member 6 HsT17432/MADH6/MADH7
SMAD7 SMAD family member 7 CRCS3/FLJ16482/MADH7/MADH8
SNAI1 Snail homolog 1 (Drosophila) SLUGH2/SNA/SNAH/SNAIL/SNAIL1/dJ710H13.1
SP1 Sp1 transcription factor -
STAT1 Signal transducer and activator of transcription 1, 91kDa DKFZp686B04100/ISGF-3/STAT91
STAT6 Signal transducer and activator of transcription 6, interleukin-4 induced D12S1644/IL-4-STAT/STAT6B/STAT6C
TGFB1 Transforming growth factor, beta 1 CED/DPD1/LAP/TGFB/TGFbeta
TGFB2 Transforming growth factor, beta 2 MGC116892/TGF-beta2
TGFB3 Transforming growth factor, beta 3 ARVD/FLJ16571/TGF-beta3
TGFBR1 Transforming growth factor, beta receptor 1 AAT5/ACVRLK4/ALK-5/ALK5/LDS1A/LDS2A/SKR4/TGFR-1
TGFBR2 Transforming growth factor, beta receptor II (70/80kDa) AAT3/FAA3/LDS1B/LDS2B/MFS2/RIIC/TAAD2/TGFR-2/TGFbeta-RII
TGIF1 TGFB-induced factor homeobox 1 HPE4/MGC39747/MGC5066/TGIF
THBS1 Thrombospondin 1 THBS/THBS-1/TSP/TSP-1/TSP1
THBS2 Thrombospondin 2 TSP2
TIMP1 TIMP metallopeptidase inhibitor 1 CLGI/EPA/EPO/FLJ90373/HCI/TIMP
TIMP2 TIMP metallopeptidase inhibitor 2 CSC-21K
TIMP3 TIMP metallopeptidase inhibitor 3 HSMRK222/K222/K222TA2/SFD
TIMP4 TIMP metallopeptidase inhibitor 4 -
TNF Tumor necrosis factor DIF/TNF-alpha/TNFA/TNFSF2
VEGFA Vascular endothelial growth factor A MGC70609/MVCD1/VEGF/VPF
B2M Beta-2-microglobulin
HPRT1 Hypoxanthine phosphoribosyltransferase 1 HGPRT/HPRT
RPL13A Ribosomal protein L13a L13A/TSTA1
GAPDH Glyceraldehyde-3-phosphate dehydrogenase G3PD/GAPD/MGC88685
ACTB Actin, beta PS1TP5BP1
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*

Shaded = Housekeeping genes

Table 2. qRT-PCR array data comparing human corneal fibroblasts (HCF) stimulated with either TGF-β1 (T1) or -β3 (T3) to Control.

Gene Symbol T1 vs Control T3 vs Control
4 hours 3 days 4 hours 3 days
Fold Regulation P Fold Regulation P Fold Regulation P Fold Regulation P
AGT 2.4051 ** 2.7865 **
BCL2 2.3968 *** 2.1363 ***
CAV1 (-4.6375) *** (-2.5319) *** (-3.1986) ***
CCL11 5.1615 ** (-8.187) *** 6.3252 ** (-8.7147) ***
CCL2 2.0108 *** (-8.604) *** (-4.0731) **
COL1A2 2.2983 ***
COL3A1 2.1019 ** (-2.0082) ** 3.8563 ***
CTGF 10.2398 *** 18.4856 * 9.0303 *** 24.3563 *
DCN (-4.7623) *** (-2.6204) ***
EDN1 25.7728 *** 7.2518 *** 26.0602 *** 10.4196 **
FASLG 6.1807 *** 4.9983 **
GREM1 2.0721 *** 2.3891 ***
HGF (-73.5167) *** (-2.2872) *** (-55.9388) ***
IL13RA2 (-7.586) *** (-4.7047) ***
IL1A 2.6502 *** 2.055 ***
IL1B (-3.6092) ***
INHBE (-2.8558) *** 127.2628 *** 158.9272 ***
ITGA1 2.1852 *** 3.7883 ***
ITGA2 2.372 ***
ITGAV 4.9512 *** 3.8445 *** 3.7405 **
ITGB3 2.3775 *** (-3.1167) *
ITGB5 2.6182 ***
ITGB6 61.8676 *** 16.1486 ** 53.8837 *** 32.9884 **
JUN (-2.5501) *** (-2.9729) ***
LOX 3.0455 *** 4.266 ***
MMP1 4.4314 *** (-3.1203) ** 4.3483 ***
MMP13 3.8984 **
MMP2 2.1658 **
MMP3 (-3.9816) **
PDGFA 5.3127 *** 8.6638 *** 3.8712 *** 9.5924 ***
PLAT 2.4415 ***
PLAU 2.3475 *** (-15.8345) *** (-4.9478) **
SERPINA1 3.4929 *** 2.3907 *
SERPINE1 4.3352 *** 5.6503 *** 3.8649 *** 7.1464 ***
SERPINH1 (-2.0499) *** (-2.8243) ***
SMAD3 (-10.0329) *** (-2.6961) *** (-7.3723) ***
SMAD4 2.4107 ***
SMAD6 (-3.3571) ** (-3.6273) ***
SMAD7 3.4809 *** 3.2603 * 5.7846 *
SNAI1 2.1212 **
TGFB2 2.1523 **
THBS1 4.4314 *** 2.5393 *** 4.265 **
TIMP3 2.9542 **
VEGFA 7.5655 *** 2.5997 * 7.9234 *** 3.6056 ***
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Upregulated (black) or downregulated (red) genes that were statistically significant in HCF when stimulated with either T1 or T3 as compared with Control samples at 4 hours and 3 day. By 4 hours with T1 stimulation, there were 25 genes that were significantly upregulated and 4 downregulated as compared with Control. By 3 days, the upregulated quantity decreased to 10 genes and the downregulated increased to 12 genes. With T3 by 4 hours, there were 16 genes significantly upregulated and 7 genes downregulated, and by 3 days there were 19 genes upregulated and 8 genes downregulated.

*

P<0.05,

**

P<0.01 and

***

P<0.001

Only genes that were altered by 2-fold or greater are included.

One of the goals of our study was to determine if differential alterations in gene expression could be determined between T1- and T3-stimulated samples as compared with control. As seen in Table 2, many of the genes showed the same regulation by both T1 and T3. The 3 genes that were most highly stimulated by both T1 and T3 as compared with control at 4 hours were ITGB6, END1, and CTGF, while at 3 days they were INHBE, ITGB6, and CTGF. ITGB6 and CTGF mRNA levels were maintained at both time points. In Table 3, the effect of T3 was compared with T1. Although both growth factors stimulated numerous changes, at 4 hours only Smad7 mRNA levels were significantly different (-2.0 fold) between the two groups. At the longer time point (3 days) an increased number of changes were observed, MMP1, PLAU, IL1B (Interleukin-1 beta), ITGA1, and THBS1 were all significantly higher in T3 samples as compared with T1 (Table 3).

Table 3. qRT-PCR array data comparing human corneal fibroblasts (HCF) stimulated with TGF-β3 (T3) to those stimulated with TGF-β1 (T1).

T3 vs T1
Gene Symbol 4 hours 3 days
Fold Regulation P Fold Regulation P
IL1B 2.7155 **
ITGA1 2.3948 **
MMP1 3.2736 *
PLAU 3.2003 *
SMAD7 (-2.0284) **
THBS1 2.339 **
Open in a new tab

Statistically significant regulation of genes that were either up- or downregulated in HCF when stimulated with T3 as compared to those stimulated with T1 at 4 hours and 3 days. By 4 hours, only Smad7 appeared in T3 samples to have a statistically significant decrease in mRNA as compared with T1; however, by 3 days, 5 genes were statistically significant in T3 samples compared with T1, and there were no decreases of note.

*

P<0.05,

**

P<0.01 and

***

P<0.001

Only genes that were altered by 2-fold or greater are included.

3.3. Protein expression

From the qRT-PCR data, we found that compared to control, mRNA levels of many fibrosis-related genes were significantly altered by 4-hour or 3-day treatment with T1 or T3. To determine if protein levels reflected the changes seen in mRNA levels, we analyzed by western blot the protein product of two genes that were differentially regulated, THBS1 and Smad7, and two genes that were similarly regulated, Collagen I and Smad3. Only 3-day protein samples were analyzed due to insufficient time for the 4-hour samples to be substantially altered. As shown in Figure 2, by 3-days exposure to T1 or T3, Smad7 was significantly decreased by T1 (p<0.001); however, T3 had no significant effect. THBS1 was significantly increased by T3, 2.5-fold higher than control (p<0.05); however, T1 had no significant effect. Finally, Collagen I was significantly increased (p<0.05) and Smad3 was dramatically inhibited (p<0.001). These data agree with the qRT-PCR data.

Figure 2. Western blot analysis of selected genes.

Figure 2

Open in a new tab

Selected genes of interest (type I collagen [Col I], Smad3, Smad7, and thrombospondin-1 [THBS1]) were examined by western blot to determine if the protein reflected the changes seen in the qRT-PCR data at 3 days. (A) Graph showing Average Fold Enhancement of samples treated either with Vitamin C only (Control, VitC), TGF-β1 (T1) or TGF-β3 (T3). (*P<0.05 and ***P<0.001) (B) Representative western blots for the data presented in (A).

4. Discussion

TGF-β plays a critical role in wound healing and the pathogenesis of fibrosis (or scarring). Therefore, determining how TGF-β regulates the expression of fibrosis-related genes is essential to understanding the mechanisms of these processes. Three isoforms of TGF-β have been identified in mammals—T1, T2, and T3. Previous studies (Karamichos et al., 2011) have shown that the addition of T1 or T2 to our 3D HCF culture resulted in a fibrotic matrix, including increased expression of type III collagen and SMA. When T3 was added to these cultures, an enhanced matrix was produced compared to control, with relative thickness similar to that of the T1-treated construct; however, there was no evidence of fibrotic markers. Identifying the genes regulated by T1 and T3 in HCF may help us to understand the mechanism of corneal fibrosis associated with TGF-β. Here we undertook the molecular comparison of fibrosis-related gene expression in HCF exposed to T1 or T3 in the presence of VitC to gain further insights into the regulation and roles of these two isoforms on the fibrotic response. We choose to examine 2D cultures rather than 3D in order to reduce variability and increase sample amounts. In our initial studies, mRNA levels of 84 key genes involved in human fibrosis were tested using qRT-PCR array technology. After which, Western blot was performed to compare the protein expression of several genes that showed dramatic changes of mRNA. Two time points, 4 hours and 3 days of TGF-β treatment, were chosen in the present study to examine both rapid and slow responses.

Surprisingly, although many genes were regulated by both T1 and T3 at the early 4-hour time point, only one gene (Smad7, an I-Smad) appeared to be differentially regulated. At 4 hours as compared to control, Smad7 mRNA expression was upregulated by T1 (∼3 fold) and maintained at that level through at least 3 days. With T3 stimulation, Smad7 mRNA did not show any significant increase by 4 hours; however, by 3 days, Smad7 mRNA levels were dramatically upregulated (∼6 fold). The western blot data (Fig. 2) agrees with the qRT-PCR data in that there is a difference in Smad7 expression in HCF stimulated with T1 and T3. At 3 days, Smad7 protein expression significantly decreased with T1, but significantly increased with T3; however, the changes in mRNA and protein levels due to T1 stimulation of the HCF were not consistent. This may be due to the fact that Smad7 is known to be an intracellular antagonist of TGF-beta signaling and has been associated with decreased fibrosis (Chung et al., 2013; Dooley et al., 2003; Guo et al., 2007a; He et al., 2009; Liu et al., 2013; Ljubimov and Saghizadeh, 2015; Nakao et al., 1999; Saika et al., 2005). Our data suggests that T3, not T1, stimulates accumulation of a non-fibrotic matrix in HCF, at least partially, through upregulating Smad7 expression.

Smad3, an R-Smad, has been observed to play a critical role as a mediator of fibrotic response in models for various tissues and organs both in vitro and in vivo (Flanders, 2004). Our qRT-PCR data shows that compared to control, HCF Smad3 mRNA started to decrease by 4 hours of T1 treatment and was significantly downregulated (10 fold) by 3 days. The decrease in Smad3 mRNA caused by T3, however, was more significant earlier on. By 4 hours of T3 exposure, Smad3 mRNA was significantly decreased by 2.7 fold and continued to decrease to 7.4 by 3 days. The inhibitory effect of T1 and T3 on Smad3 expression was confirmed by examining protein levels (Fig. 2). Our western blot results showed that Smad3 protein expression was at an almost non-detectable level after HCF were exposed to T1 or T3 for 3 days, which agrees with previous studies (Poncelet et al., 2007; Wang et al., 2005; Yanagisawa et al., 1998). Yanagisawa et al. showed that the downregulation of Smad3 mRNA in human lung epithelial cells was observed as early as 4 hours after exposure to TGF-β, with a 50% reduction at 9.3 hours (Yanagisawa et al., 1998). Other studies showed that Smad3 protein levels in tubular epithelial cells (Poncelet et al., 2007) and rat bone marrow-derived mesenchymal stem cells (Wang et al., 2005) were unaffected by T1 at early time points (less than 24 hours), but decreased markedly after 24 hours. Constitutive expression (Yanagisawa et al., 1998) or overexpression of Smad3 (Cao et al., 2007) in the presence of TGF-β has been shown to induce apoptosis in epithelial cells, and the decline in Smad3 levels has been shown to help the cells avoid apoptotic death (Poncelet et al., 2007). Indeed, in our 3D construct model, after 4 weeks of exposure to TGF-β isoforms, HCF cell number was significantly increased compare to control (Karamichos et al., 2011). We speculate that with T1 or T3 stimulation, HCF proliferate and escape from apoptosis by lowering the Smad3 expression.

At the 3-day time point, we observed five genes that were differentially expressed between the T1 and T3 groups. Although none of these genes explain the differential affect of T1 and T3 on fibrosis, they do demonstrate that the two isoforms do have differing affects. Interestingly, all of the five genes MMP1 (matrix metalloproteinase 1), PLAU (plasminogen activator, urokinase), IL1B (Interleukin-1 beta), ITGA1 (integrin alpha 1) and THBS1 either interact with or are part of the extracellular matrix. This would suggest that the differential effect of T1 and T3 might involve interaction with matrix. Since, our previous studies (Blanco-Mezquita et al., 2013; Matsuba et al., 2011; Zieske et al., 2001) demonstrated that THBS1 was upregulated in wounded cornea and healing in THBS1-deficient corneas was impaired, we further examined this protein. THBS1 is a multifunctional matrix glycoprotein that plays an important role in the healing response. Here we give direct evidence showing that both T1 and T3 upregulate THBS1 expression in HCF. Our qRT-PCR results showed that THBS1 mRNA was elevated at 4-hour treatment with both T1 and T3, indicating an early THBS1 transcriptional response to both T1 and T3 stimulation. After 3 days, THBS1 mRNA decreased to normal in T1 but continued to increase in T3, showing significant difference between T1 and T3 treated cells, which were in agreement with the western blot results. Although peak expression of THBS1 in HCF was observed at 24 hours of treatment with T1 (unpublished observations), in current studies (Fig. 2), the amount of THBS1 protein was not significantly higher than control after 3 days; however, T3 dramatically stimulated THBS1 protein expression by 3 days. It remains to be elucidated why T1 and T3 have such a different regulation on THBS1 expression in HCF.

One of our original observations that stimulated our project was the finding that in long-term HCF cultures (4 weeks), T1 stimulated fibrosis and SMA expression, while T3 did not. We anticipated that our current study would agree with this finding. With IF, we found that in short-term cultures (Fig. 1), there was a greater number of SMA-positive cells in the T1-treated HCF than the T3, which agrees with the long-term culture data; however, both T1 and T3 had significantly more SMA-positive cells than controls. With our array data, we also observed that T1 and T3 both stimulated SMA-gene expression at short time points. These data are in agreement with Agarwal and Wang (Agarwal and Wang, 2005) who found in ligament fibroblasts that T1 and T3 had similar effects at short time points. The array data may simply reflect one of the pitfalls of gene arrays in that not all probes may be equally effective, and that gene arrays should only be considered a starting point to direct the initiation of studies. However, we have also observed in our laboratory that SMA mRNA levels do not always reflect protein level and the fact that with short-term IF cultures, the data agreed somewhat with the array data. Therefore, these findings may suggest that the differential effect of T1 and T3 is not a simple matter of relative level of SMA mRNA, but in fact T1 and T3's effect may be slow to develop and complex. This may in part help explain why T3 has shown to be beneficial in some animal models, but has failed clinical trials to improve scarring (Akhurst and Hata, 2012; Ferguson et al., 2009; Finnson et al., 2013).

In conclusion, the alteration of fibrosis-related molecular expression in HCF caused by T1 or T3 stimulation was compared using qRT-PCR. Based on the PCR results, four genes were chosen to test the protein level change. Although none of the genes totally explain the differential effect of T1 and T3 on fibrosis, the results are novel in that they do identify gene targets that are differentially regulated. We found that compared to T1, T3 may stimulate non-fibrotic matrix by upregulating Smad7 and THBS1. Further work is needed to clarify why T1 and T3 have different regulation on the other genes and how they may differentially regulate fibrosis.

Highlights.

  • TGF-β1 and -β3 have similar effects on the expression of fibrotic genes at 4 hours.

  • At 3 days, there were more differentially expressed genes between TGF-β1 and -β3.

  • Both TGF-β1 and -β3 decreased HCF Smad3 mRNA at both time points.

  • TGF-β3 may stimulate non-fibrotic matrix by upregulating Smad7 and THBS1.

Acknowledgments

This study was funded by grants from National Institute of Health (EY03790 and EY005665) and Department of Defense (W81XWH-11-1-0477). Authors have no commercial interests to disclose. Data in this manuscript has been previously presented at ARVO 2013 as a poster.

Footnotes

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Contributor Information

Audrey E. K. Hutcheon, Email: Audrey_Hutcheon@meei.harvard.edu.

James D. Zieske, Email: James_Zieske@meei.harvard.edu.

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