Ultraviolet B-induced expression of amphiregulin and growth differentiation factor 15 in human lens epithelial cells.
OBJECTIVE
Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis.


METHODS
SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10-80 mJ/cm²) and checked for viability. An irradiation condition of 30 mJ/cm² was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT-PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins.


RESULTS
Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT-PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated ³H-thymidine and ³H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate ³H-thymidine incorporation at any concentration tested, but significantly stimulated ³H-leucine uptake. RT-PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor β receptors (TGFBR1 and TGFBR2) mRNAs.


CONCLUSIONS
These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.
Department of Ophthalmology, Kanazawa Medical University School of Medicine, Uchinada, Ishikawa, Japan.
Hiromi
H
Osada
Yoshino
Y
Yoshitake
Takayuki
T
Ikeda
Yasuhito
Y
Ishigaki
Takanobu
T
Takata
Naohisa
N
Tomosugi
Hiroshi
H
Sasaki
Hideto
H
Yonekura
D066258:Amphiregulin; D002386:Cataract / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D066255:EGF Family of Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology* / Q000528:radiation effects; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D055436:Growth Differentiation Factor 15 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D007908:Lens, Crystalline / Q000166:cytology* / Q000528:radiation effects; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014466:Ultraviolet Rays
C587848:AREG protein, human; D066258:Amphiregulin; D017931:DNA Primers; D066255:EGF Family of Proteins; C526140:GDF15 protein, human; D006023:Glycoproteins; D055436:Growth Differentiation Factor 15; D036341:Intercellular Signaling Peptides and Proteins
Br J Ophthalmol. 1999 Feb;83(2):225-30
10396203
Int J Cancer. 2009 Feb 1;124(3):531-9
19003995
J Invest Dermatol. 2006 Apr;126(4):732-9
16470170
Arch Biochem Biophys. 1988 Jun;263(2):437-46
2454080
J Clin Invest. 1997 Sep 1;100(5):1271-81
9276746
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2681-7
9856778
Br J Ophthalmol. 1998 Aug;82(8):945-52
9828783
Circ Res. 2006 Feb 17;98(3):351-60
16397141
FASEB J. 1995 Sep;9(12):1173-82
7672510
Exp Eye Res. 2000 Dec;71(6):609-18
11095913
Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11514-9
9326641
Jpn J Ophthalmol. 1993;37(3):310-7
8295370
J Membr Biol. 1996 Mar;150(1):89-103
8699483
FEBS Lett. 2009 Mar 4;583(5):933-7
19302795
Nat Med. 2007 Nov;13(11):1333-40
17982462
Exp Eye Res. 1998 Nov;67(5):577-85
9878220
J Biol Chem. 1998 May 29;273(22):13760-7
9593718
Cell Tissue Res. 2004 Nov;318(2):325-33
15459768
J Biol Chem. 1996 Jul 26;271(30):17927-31
8663535
Science. 1993 Nov 12;262(5136):1032-4
17782050
Biochim Biophys Acta. 1997 Oct 9;1354(1):40-4
9375789
Cell Signal. 2009 Feb;21(2):212-9
18951974
Ophthalmic Epidemiol. 2002 Apr;9(2):83-95
11821974
Arch Ophthalmol. 1989 Mar;107(3):369-72
2923558
Circ Res. 2006 Feb 17;98(3):342-50
16397142
Science. 1989 Feb 24;243(4894 Pt 1):1074-6
2466334
Invest Ophthalmol Vis Sci. 2000 Jan;41(1):204-12
10634622
Hepatology. 2008 Oct;48(4):1251-61
18634036
Curr Eye Res. 1995 Jan;14(1):71-8
7720407
Endocrinology. 2009 Apr;150(4):1688-96
19074584
Development. 2005 Sep;132(17):3923-33
16079154
Differentiation. 1981;19(3):134-53
7030840
Invest Ophthalmol Vis Sci. 1996 Aug;37(9):1720-3
8759338
Invest Ophthalmol Vis Sci. 2000 Apr;41(5):1130-41
10752951
Ageing Res Rev. 2002 Sep;1(4):705-20
12208239
Curr Eye Res. 1999 Mar;18(3):224-30
10342377
J Biol Chem. 2005 May 13;280(19):19012-20
15753092
Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):109-14
10618379
Gene. 1997 Dec 5;203(1):17-26
9426002
Free Radic Biol Med. 2004 Apr 1;36(7):881-96
15019973
Eye (Lond). 2005 Oct;19(10):1133-5
16304595
J Invest Dermatol. 2008 Jun;128(6):1365-74
18049451
Br J Dermatol. 2007 Nov;157(5):874-87
17711532