Identification of Transforming Growth Factor ... - Semantic Scholar
Identification of Transforming Growth Factor-/? Expressed in Cultured Human Retinal Pigment Epithelial Cells Hidenobu Tanihara, Munenori Yoshida, Miho Matsumoto, and Nagahisa Yoshimura
Purpose. This study examined whether retinal pigment epithelial (RPE) cells have the capacity to express transforming growth factor-/? (TGF-/3). Also examined were TGF-j8 isoform genes expressed in the RPE cells. Methods. Complementary DNA (cDNA) was generated from polyA+ RNA extracted from human RPE cells in culture, and polymerase chain reaction (PCR) using degenerate and specific primers of the known TGF-j8 isoforms was carried out by using the cDNAs as templates. Sequencing and Southern blot analysis of the PCR products, and Northern blot analysis were performed to identify which isoforms are expressed in RPE cells. Results. PCR using degenerate primers showed that most of the amplified sequences were derived from TGF-/32, although TGF-/31 expression was shown by using specific primers. Northern blot analyses confirmed not only the expression of the IGF-/3 gene, but also suggested that: alternative splicing of TGF-/3 mRNA in the RPE cells occurred. Conclusion. Cultured human RPE cells expressed TGF-j3.1 and /32 genes, whereas gene expression of TGF-j83 was not confirmed. Our data suggest that RPE cells are intraocular origin of TGF-/3 production. Invest Ophthalmol Vis Sci. 1993;34:413-419.
Jbvecent studies have revealed that RPE cells release some growth factors, such as basic fibroblast growth factor, insulin-like growth factor, and platelet-derived growth factor.1"4 Also, cultured RPE cells have been shown to have the capacity to produce cytokines such as interleukin-6, interleukin-8, and macrophage colony stimulating factor in response to other cytokines.5"8 Thus, it is possible that RPE cells contribute to the development of ocular diseases by releasing chemical modulators. Transforming growth factor-/? (TGF-j8) is a wellknown cytokine that elicits numerous changes in cellu-
lar behavior, including proliferation, in vivo angiogenesis, and deposition of extracellular matrix.9 The TGFfi gene family is composed of at least five members (TGF-/31 through 5). Among these, three (TGF-01, 2, and 3) are known to be expressed in mammalian tissues,10"12 whereas the other two (TGF-/34 and 5) are expressed only in nonmammalian tissues. The majority of TGF-/3 is secreted as inactive complexes of high molecular weight.13 In the present report, we will show that cultured human RPE cells express TGF-/3 and TGF-/3 binding protein genes. We also will demonstrate that the isoforms of TGF-/3 expressed by RPE cells are TGF-01 and TGF-/32.
From the Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research (04454441) from the Ministry of Education, Science and Culture of the Japanese Government. Submitted for publication: May 13, 1992; accepted July 23, 1992. Proprietary interest category: N. Reprint requests: Dr. Nagahisa Yoshimura, Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
Cell Culture RPE cells were obtained from aborted fetuses of 1722 wk gestation, as described previously.14 The research was approved by the institutional human experi-
lnvesligalivc Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2 Copyright © Association for Research in Vision and Ophthalmology
413
MATERIALS AND METHODS
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
414
mental committee, and in the author's opinion, methods for securing human tissue were humane, included proper consent and approval, and complied with the tenets of the Declaration of Helsinki. Briefly, after washing with Ca2+ and Mg2+ free phosphate buffered saline (PBS), the anterior segment and the vitreous were removed and the sensory retina was discarded. The remaining eye cup was cut into quadrants. Sheets of RPE cells were peeled off from the choroid, recovered by brief centrifugation (800 rpm), and transferred to a 60 mm culture dish containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin G (100 IU/ml), streptomycin (100 jug/ml), and amphotericin B (0.25 mg/ml). RPE cells were cultured in an incubator with a humidified atmosphere of 5% CO2 and 95% air at 37°C. When RPE cells were cultured to near confluency, the cells were washed with PBS solution, incubated for 10 min at 37°C with Ca2+ and Mg2+ free Hank's solution containing 0.02% ethylenediaminetetraacetic acid
(EDTA) and 0.25% trypsin, and subcultured into new culture dishes with a ratio of 1:3. Three to four days after subculture, at the stage of near confluency, RPE cells were harvested. Cells from the third to sixth passages were used in the experiments. In this culture condition, RPE cells were not fully pigmented. About 60 fig of total RNA or 1.5 ^g of polyA+, RNA was prepared from one 100 mm culture dish.
Polymerase Chain Reaction PolyA+ RNA was prepared from the cultured human RPE cells by using Micro-Fast Track (Invitrogen, San Diego, CA). First strand complementary DNA (cDNA) was prepared from the polyA+ RNA using the FirstStrand cDNA synthesis kit (Pharmacia-LKB, Uppsala, Sweden). The primers were synthesized by an oligonucleotide synthesizer (Model 392; Applied Biophysics, Foster City, CA). We chose two highly conserved amino acid sequences by comparing various TGF-/3 sequences and used these to make degenerate primers
l. Amino Acid Sequence Similarity Between the Human TGF-tfl, (32 and #3 Precursors
TABLE
0] 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03
MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRK MH-YCVLSAFLILHLVTVALSLSTCSTLDMDQFMRK MHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKK RIEAIRGQILSKLRLASPP SQGEVPPGPLPEAVLALYNSTR RIEAIRGQILSKLKLTSPPEDYPEPEEVPP EVISIYNSTR RVEAIRGQILSKLRLTSPPEPTVMTH-yP YQVLALYNSTR D R-VAGESAEPEPEPEADYYAKEVTRVLMV---ETH DLLQE-KASRRAAACERERSDE EYYAKEVYKIDMPPFFPSE ELLEEMHGERE-EGC-TQENTES---EYYAKEIHKFDMIQGLAEH NEIYDKFKQSTHSIYMFF NTSELREAVPEPVLLSRAELR NAIPPTFYRPYFRIVRFDVSAMEKNAS NLVKAEFR NELAVCPKGITSKVFRF NVSSVEKNRT---NLFRAEFR LLRL-KLK---VEQHVELYQKYSNNSW RYLSNRLLAPSDS VFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAE VLRVPNPSSKRNEQRIELFQILRPDEHIAK-QRYIGGKNLPTRGT PEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC GEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIP AEWLSFpyTDTVREWLLRRESNLGLEISIHCPCHTFQP-N-GDIL --DSRDNTLQVDINGFTTGR---RGDIATIHGM -NRPFLL NKSEELEARFAGIDGTSTYTS---GDQKTIKSTRKKNSGKTPHLL --ENIHEVMEIKFKGVDNEDDHFRGDLGRLKK--QK-DHHNPHLI
01 02 03 01 02 03 01 02 03
LMATPLERAQH--LQSS-RHRRALDTNYCFSSTEKNCCVRQLYID LMLLPSYRLES--QQTNRRKKRALDAAYCFRNVQDNCCLRPLYID LMMIPPHRLDNPGQGGQ-RKKRALDTNYCFRNLEENCCVRPLYID FRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQH FKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTI FRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTyLGLYNTL NPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS NPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS NPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS
I "
Dashes are introduced for alignment. Identical amino acids are marked by dots. Highly conserved amino acid sequences are underlined. The arrow indicates the start of the mature TGF-/8 sequences.
TGF-/? Expression in RPE Cells
415
that contained all the possible nucleotide sequences (Tables 1 and 2). Specific primers for TGF-/31, 2, and 3, and TGF-/31 binding protein were synthesized by choosing specific nucleotide sequences from the previously published sequences of human TGF-/31 through 3 10 - 12 and TGF-/31 binding protein13 (Table 2). Primers were designed so that introns were included in expected genomic sequences. Possible amplification of contaminating genomic DNA thus could be detected by the larger size of the polymerase chain reaction (PCR) products. PCR was carried out by the method of Saiki et al,15 with a slight modification.16 The following conditions were used: denaturation, at 94°C for 1.5 min; annealing, 55 or 65°C for 2 min; polymerization, 72°C for 3 min. The reaction was initiated by adding 2 U of Taq polymerase, after which 35 cycles were carried out. Taq DNA polymerase and reagents for PCR experiments were obtained from Perkin-Elmer Cetus (Norwalk, CT). After the reaction, the products were separated by 1.5% agarose gel electrophoresis, and bands of the expected length were extracted. The resulting DNAs were subcloned into the pBluescript II vector (Stratagene, La Jolla, CA). Before subcloning, the vector was treated by Eco RVand the T vectors were made by Taq polymerase and deoxythymidine triphosphate.17 Sequencing of the PCR Products To confirm that PCR products were derived from nucleotide sequences of TGF-/3 isoforms, sequencing of the subcloned DNAs was carried out according to the TABLE 2.
dideoxynucleotide chain termination method18 using a Sequenase ver. 2.0 DNA sequencing kit (United States Biochemicals, Cleveland, OH). Double-strand template DNA was denatured by alkaline treatment, and the sequencing reaction was initiated by adding T3 or T7 primers. Southern and Northern Blot Analysis For Southern blot analysis, 10 /xl of PCR products were separated by 1.5% agarose gel electrophoresis and transferred to a nylon filter (Hybond-N+; Amersham, Buckinghamshire, UK) by the capillary transfer method with 20X standard saline citrate (SSC). The DNA was fixed to the filter by baking at 80°C for 2 hr. DNA fragments corresponding to the target sequences were labeled by an enhanced chemoluminescence gene detection kit (Amersham). Probes used were a 336 bp DNA fragment for human TGF-j81 and a 384 bp DNA fragment for TGF-/32. After hybridization, the filters were washed with the washing buffer (urea 360 g, sodium dodecyl sulfate 4 g, 20X SSC 25 ml/1) twice at 42°C, then with 2X SSC twice at room temperature. PolyA+ RNA preparations were electrophoresed in a 0.8% agarose gel under denaturing conditions and transferred onto a nylon filter using the capillary method. Northern blot analysis was performed according to the method of Thomas.19 Probes used were a 2.1 kbp pair DNA fragment for TGF-/51 that encodes the complete coding and partial flanking sequence of TGF-/31 and the 384 bp DNA fragment for TGF-/32. DNA fragments were labeled with deoxycytidine 5'«-
Primers for Polymerase Chain Reaction
(J) Amino acid and corresponding nucleotide sequences for degenerate primers. TGF-jSs (mixed oligonucleotides) sense anti-sense
(EWLSFD) 5' (DLGWKW) 5'
CAXTGGYTZTCZTTYGA CAYTTCCAZCCZAXXTC
(2) Nucleotide sequences for specific primers. TGF-/31 (expected length, 336 bp) sense anti-sense TGF-/32 (384 bp) sense anti-sense TGF-/33 (300 bp) sense anti-sense TGF-/3 binding protein (467 bp) sense anti-sense
5' 5'
GCCCTGGACACCAACTATTGC GCTGCACTTGCAGGAGCGCAC
5' 5'
TACAGACCCTACTTCAG AAATCTTGCTTCTAGTT
5' 5'
GCACTTGCAAAGGGCTC TTGGCATAGTATTCCGA
5' 5'
TGAGTAACCACACTGGC AACCTGATGTATCTGGA
Amino acid sequences were chosen by comparing human TGF-/31 to 3. 10 " 12 The nucleolide sequence is as follows: A, deoxyadenosine; C, deoxyguanosine; C, deoxycylidine; T, deoxythymidine; X, either A or G; Y, either C or T; Z, either A, G, C, or T.
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
416 32
P triphosphate (Amersham Japan, Tokyo, Japan) by Multiprime DNA labeling system (Amersham). Hybridization was carried out at 42DC in the hybridization buffer containing 50% fonnamide. The final wash was with 0.1X SSC that contained 0.5% sodium dodecyl sulfate at 60°C for 20 min. RESULTS
B 1
2
3
4
1 2
3
4
4
Identification of TGF-/? Isoforms by PCR Using Degenerate Primers PCR using degenerate primers was applied to isolate cDNAs that correspond to the known TGF-jS isoforms or possible new isoforms expressed in human RPE cells. To prepare degenerate primers longer than 17 or 18 nucleotides, five highly conserved amino acid sequences were selected by comparing various human TGF-jS isoforms: EAIRGQILSKL, EWLSFD, DLGWKW, HEPKGY, and EQLSNM (Table 1). Among these sequences, two pairs (EVVLSFD and DLGWKW; HEPKGY and EQLSNM) were chosen for the PCR experiments. Figure. 1 indicates that the products of the expected length were detected from PCR experiments using degenerate primers, (EWLSFD and DLGWKW). The resulting PCR products of the expected length were subctoned into the pBltiescript II vector. Eighteen clones from the PCR experiments were sequenced. Thirteen (72%) out of
-310
FICURE l. Reaction products of PCR experiments using degenerate primers for TGF-/J.
FIGURE 2. Southern blot analysis of PCR products. PCR product (10 M') was separated by agarose gel eteci rophoresis and transferred onto a nitrocellulose filter. The filter was hybridized with the DNA fragment labeled by enhanced chemoluminescence. Labeled probe A, TGF-/32; B, TCF-/3.1. Lane 1, products from PCR using degenerate primers. Lane 2, PCR products for TGF-0 binding proiein. Lane 3, PCR products for TGF-03. Lane 4, PCR products for TGF-02. Lane 5, PCR products for TGF-jSl.
the 18 clones were found to encode nucleotide sequences of human TGF-/32. The remaining five clones (28%) had no amino acid sequences homologous to the known TGF-/3s. These results suggest that human RPE cells primarily express genes of TGF-/32. By Southern blot analysis, one positive band was detected from the PCR products using labeled TGF-/32 DNA fragments as a probe (Fig. 2). Subcloning and subsequent sequencing of the PCR products using another pair of primers (HEPKGY and EQLSNM) revealed that most of the products were derived from other than human TGF-jSs. Identification of TGF-/3 Isoforms by PCR Using Specific Primers To confirm the expression of TGF-/31, 2, and 3, we performed additional PCR experiments using primers encoding the specific nucleotide sequences of TGF/31, 2, and 3. Figure 3 indicates that the PCR products of the expected length were detected for TGF-/31 and 2, but not for TGF-/23, although very faint bands appeared. We carried out one more PCR experiment using the PCR products as the template and tried to discover possible expression of TGF-/33. However, we could not efficiently amplify PCR products of the appropriate length by additional PCR experiments and failed to subclone the products. Thus, expression of TGF-j83 gene was not confirmed by our analysis. We isolated and subcloned PCR products of TGF-/31 and 2 into the pBluescript II vector and sequenced. Again, however, all nucleotide sequences of subcloned PCR products from this experiment revealed sequences
417
TGF-0 Expression in RPE Cells
1 2
3
PCR products, subcloning and subsequent sequencing were performed. Sequencing of the subcloned PCR products revealed that the amplified sequences were derived from TGF-jS binding protein. This indicates that human RPE cells express messages not only of TGF-jS, but also of the TGF-,3 binding protein.
DISCUSSION
- 603 bp -310
FIGURE 3. Reaction products from PCR experiments using specific primers. Lane 1, PCR products for TGF-/33. Lane 2, PCR products for TGF-02. Lane 3, PCR products for TGF-01.
TGF-jS is a cytokine that has been implicated in a number of ocular diseases. For example, a higher concentration of TGF-/? in the vitreous aspirates from eyes with proliferative vitreoretinopathy than from those with nonproliferative retinal detachment has been reported, suggesting important roles of TGF-0 in the process of intraocular proliferation of RPE and other types of the cells.20 TGF-jS induced modification of RPE cell behaviors to growth factors and RPE-mediated gel contraction also have been reported,2'122 although a controversial report also was published.23 This cytokine is known to be widely distributed in the ocular tissues. Also reported has been the recognition of cell growth modulators by anti-TGF-/? antibodies in the aqueous humor and vitreous.20'24'25 Ciliary body epithelial cells embryologically related to the RPE reportedly produce TGF-/31 and 02.26 Also, TGF-/3 im-
A
B
that corresponded to TGF-/31 and 2. Southern blot analysis confirmed that this band was derived from TGF-0] and 2 (Fig. 2). Expression of TGF-jff mRNAs The expression of mRNAs for the TGF-/31 and 2 in the RPE cells was examined by Northern blot analysis. The message for TGF-/31 was detected and the size was 2.5 kbp. The message for TGF-/31 detected by the Northern blot analysis was a single band even when we used DNA fragments that encode the complete coding sequence of TGF-/3! (Fig. 4A). Messages for TGF-02 also were detected, and their sizes were about 6.5 (main band), 5.0, and 4.0 kbp (Figs. 4B, C). PCR and Subsequent Sequencing for Detecting TGF-jS Binding Protein To identify the expression of TGF-/3 binding protein mRNA, we performed PCR experiments using human RPE cDNA as the template and one pair of specific primers that encodes the molecular structure of TGF/31 binding protein.13 PCR experiments showed that the resulting product contained DNA fragments of about 470 bp in size (Fig. 5). To characterize these
kb
I
-9.49 ^7.46 -4.40 -2.37 — 135
FIGURE 4. Northern blot analysis. PolyA+ RNA of 1 jug was elect rophoresed in 0.8% agarose gel under denaturing conditions and transferred onto a nylon filler using capillary method. The final wash was with 0. IX SSC containing- 0.5% sodium dodecyl sulfale at 60°C for 20 min. Lane A, TGF-/J1. Lane B, TGF-/32, overnight exposure. Lane C, TGF-/J2, 1 wk exposure.
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
418
-603 bp -310
FIGURE 5. Reaction products of PCR experiments using specific primers for TGF-/3 binding protein.
munoreactivity was found in adult human retinal photoreceptors. 27 Although RPE cells are thought to produce the cytokine that has an autocrine mechanism to TGF-/S and causes complicated regulation of cellular behaviors,20 production or gene expression of TGF-/? by the cell has not yet been unequivocally demonstrated. Thus, the present study was designed to confirm the gene expression of TGF-/? and to identify isoforms of TGF-/3 expressed in human RPE cells in culture. In this study, we first performed PCR experiments using degenerate primers that encode common ami no acid sequences of TGF-jSl, 2, and 3, because these three isoforms are known to be expressed in mammalian tissues. This technique of PCR is becoming an increasingly popular method for cloning new members of a gene family because of the good possibility that the new members may share common features in the primary structure of the molecules. The TGF-jS superfamily is composed of many members, including TGF-jSl to 5. 28 We thought it was possible that an unknown isoform of TGF-/? was produced in the RPE cells. However, all the subcloned DNA from the PCR experiments using degenerate primers was of TGF-/32 origin. This suggests that the number of copies of cDNA with TGF-/02 origin is much larger than that of other members of the TGF-/? gene family. It is possible that a yet unidentified member of the TGF-/3 gene family is expressed in human RPE cells, but this seems highly unlikely.
To determine the expression of the known members of the TGF-jC? gene family, we performed further PCR experiments using specific primers, because even a relatively small number of cDNA copies can be amplified by this technique. In fact, the PCR experiments showed that TGF-01 also is expressed in RPE cells, but experiments that searched for TGF-/33 were negative. Subsequent sequencing of the subcloned PCR products and Southern blot analysis confirmed that the PCR products were of TGF-/31 origin. Previous studies using bioassay to detect TGF-/3 activity in intraocular fluids such as aqueous humor and vitreous aspirates20-24-25 revealed that most of the immunoreactive TGF-/3 is TGF-/32 and only a small portion is TGFj81. Our data on the preponderance of TGF-/32 gene expression are consistent with these bioassay experiments. Northern blot analysis demonstrated multiple bands for TGF-02. Multiple bands of 6.5, 5.1, and 4.1 kbp for TGF-/32 have been reported in human cell lines, and our data agree with previous reports. 10 ' 29 There are two possible explanations for multiple messages: (1) occurrence of alternative mRNA splicing;29'30 and (2) presence of a highly homologous molecule. The former hypothesis is more likely, because our PCR experiments using degenerate primers did not suggest the presence of highly homologous molecules. Most TGF-/3 is secreted as inactive complexes of high molecular weight.13 Activation of latent TGF-/5 is an important in vivo regulatory step, although little has been elucidated about this mechanism. We have shown that human RPE cells also express the TGF-01 binding protein gene that is thought to be expressed in TGF-/?-producing cells. In summary, we have demonstrated the expression of TGF-/31, TGF-02, and TGF-/3 binding protein genes in RPE cells. Further investigations on modulators produced by healthy or deteriorating RPE cells are needed to understand the pathogenesis of many ocular disorders. Key Words Northern blot analysis, polymerase chain reaction, retinal pigment epithelial cells, transforming growth factor-/?. References 1. Schweigerer L, Malerstein B, Neufeld G, Gospodarowicz D. Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells. Biochem Biophys Res Commun. 1987; 143:934-940. 2. Sternfeld MD, Robertson JE, Sahipley GD, Tsai J, Rosenbaum JT. Cultured human retinal pigment epithelial cells express basic fibroblast growth factors and ks receptors. Curr Eye Res. 1989;8:1 029-1037. 3. Ocrnt A, Fay CT, Parmelee JT. Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment, epithelium. Exp Eye Res. 1991:52:581-589.
TGF-0 Expression in RPE Cells 4. Campochiaro PA, Sugg R, Grotendorst G, Hjelmeland LM. Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media. Exp Eye Res. 1989;49:2l7-227. 5. Jaffe GJ, Peters WP, Roberts W, et al. Modulation of macrophage colony stimulating factor in cultured human retinal pigment epithelial cells. Exp Eye Res. 1992; 54:595-603. 6. Elner VM, Scales W, Elner SG, Danfort J, Kunkel SL, Stricter RM. Interleukin-6 (IL-6) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells. Exp Eye Res. 1992;54:361-368. 7. Planck SR, Dang TT, Graves D, Tara D, Ansel JC, Rosenbaum JT. Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1. Invest Ophthalmol Vis Sci. 1992;33:78-82. 8. Elner VM, Strieter RM, Elner AG, Baggiolini M, Lindley I, Kunkel S. Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigment epithelial cells. AmJ Pathol. 1990; 136:745-750. 9. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987; 105:1039-1045. 10. Derynck R, JarrettJA, Chen EY, et al. Human transforming growth factor-/? complementary DNA sequence and expression in normal and transformed cells. Nature. 1985;316:701-705. 11. de Martin R, Haendler B, Hofer-Warbinek R, et al. Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-/3 gene family. EMBO J. 1987;6:3673-3677. 12. Derynck R, Lindquist PB, Lee A, et al. A new type of transforming growth factor-/3, TGF-/33. EMBO J. 1988; 7:3737-3743. 13. Kanzaki T, Olofsson A, Moren A, et al. TGF-01 binding protein: A component of the large latent complex of TGF-/31 with multiple repeat sequences. Cell. 1990:61:1051-1061. 14. Kuriyama S, Ohuchi T, Yoshimura N, Honda Y. Growth factor-induced cytosolic calcium ion transients in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1991;32:2882-2890. 15. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of/3-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:l 350-1354. 16. Suzuki S, Huang ZS, Tanihara H. Cloning of an integrin j8 subunit exhibiting high homology with integrin 03 subunit. Proc Nad Acad Sci USA. 1990;87:53545358.
419
17. Marchuk D, Drumm M, Saulino A, Collins FS. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991; 19:1154. 18. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977; 74:5463-5467. 19. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA. 1980; 77:5201-5205. 20. Connor TB, Roberts AB, Sporn MB, et al. Correlation of fibrosis and transforming growth factor-/? type 2 levels in the eye./ Clin Invest. 1989;83:1661-1666. 21. Leschey KH, Hackett SF, Singer JH, Campochiaro PA. Growth factor responsiveness of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1990;31:839-846. 22. Raymond MC, Thompson JT. RPE-mediated collagen gel contraction. Inhibition by colchicine and stimulation by TGF-beta. Invest Ophthalmol Vis Sci. 1990; 31:1079-1086. 23. Opas M, Dziak E. Effects of TGF-beta on retinal pigmented epithelium in vitro. Exp Cell Bio I. 1989; 57:206-212. 24. Eisenstein R, Grant-Bertacchini D. Growth inhibitory activities in avascular tissues are recognized by antitransforming growth factor B antibodies. Curr Eye Res. 1991;10:157-162. 25. Cousins SW, McCabe MM, Danielpour D, Streilein JW. Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest Ophthalmol Vis Sci. 1991; 32:2201-221 1. 26. Helbig H, Kittredge KL, Coca-Prados M, Davis J, Palestine AG, Nussenblatt RB. Mammalian ciliary-body epithelial cells in culture produce transforming growth factor-beta. Graefes Arch Clin Exp Ophthalmol. 1991; 229:84-87. 27. Lutty G, Ikeda K, Chandler C, McLeod DS. Immunohistochemical localization of transforming growth factor-/3 in human photoreceptors. Curr Eye Res. 1991; 10:61-74. 28. Massague J. The transforming growth factor-/? family. Annu Rev Cell Biol. 1990;6:597-641. 29. Webb NR, Madisen L, Rose TM, Purchio AF. Structural and sequence analysis of TGF-02 cDNA clones predicts two different precursor proteins produced by alternative mRNA splicing. DNA. 1988;7:493-497. 30. Kondaiah P, Obberghen-Schilling EV, Ludwig RL, Dhar R, Sporn MB, Roberts AB. cDNA cloning of porcine transforming growth factor-/?! mRNAs. Evidence for alternate splicing and polyadenylation. J Biol Chem. 1988;263:18313-18317.
Purpose. This study examined whether retinal pigment epithelial (RPE) cells have the capacity to express transforming growth factor-/? (TGF-/3). Also examined were TGF-j8 isoform genes expressed in the RPE cells. Methods. Complementary DNA (cDNA) was generated from polyA+ RNA extracted from human RPE cells in culture, and polymerase chain reaction (PCR) using degenerate and specific primers of the known TGF-j8 isoforms was carried out by using the cDNAs as templates. Sequencing and Southern blot analysis of the PCR products, and Northern blot analysis were performed to identify which isoforms are expressed in RPE cells. Results. PCR using degenerate primers showed that most of the amplified sequences were derived from TGF-/32, although TGF-/31 expression was shown by using specific primers. Northern blot analyses confirmed not only the expression of the IGF-/3 gene, but also suggested that: alternative splicing of TGF-/3 mRNA in the RPE cells occurred. Conclusion. Cultured human RPE cells expressed TGF-j3.1 and /32 genes, whereas gene expression of TGF-j83 was not confirmed. Our data suggest that RPE cells are intraocular origin of TGF-/3 production. Invest Ophthalmol Vis Sci. 1993;34:413-419.
Jbvecent studies have revealed that RPE cells release some growth factors, such as basic fibroblast growth factor, insulin-like growth factor, and platelet-derived growth factor.1"4 Also, cultured RPE cells have been shown to have the capacity to produce cytokines such as interleukin-6, interleukin-8, and macrophage colony stimulating factor in response to other cytokines.5"8 Thus, it is possible that RPE cells contribute to the development of ocular diseases by releasing chemical modulators. Transforming growth factor-/? (TGF-j8) is a wellknown cytokine that elicits numerous changes in cellu-
lar behavior, including proliferation, in vivo angiogenesis, and deposition of extracellular matrix.9 The TGFfi gene family is composed of at least five members (TGF-/31 through 5). Among these, three (TGF-01, 2, and 3) are known to be expressed in mammalian tissues,10"12 whereas the other two (TGF-/34 and 5) are expressed only in nonmammalian tissues. The majority of TGF-/3 is secreted as inactive complexes of high molecular weight.13 In the present report, we will show that cultured human RPE cells express TGF-/3 and TGF-/3 binding protein genes. We also will demonstrate that the isoforms of TGF-/3 expressed by RPE cells are TGF-01 and TGF-/32.
From the Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto, Japan. This study was supported in part by a Grant-in-Aid for Scientific Research (04454441) from the Ministry of Education, Science and Culture of the Japanese Government. Submitted for publication: May 13, 1992; accepted July 23, 1992. Proprietary interest category: N. Reprint requests: Dr. Nagahisa Yoshimura, Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
Cell Culture RPE cells were obtained from aborted fetuses of 1722 wk gestation, as described previously.14 The research was approved by the institutional human experi-
lnvesligalivc Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2 Copyright © Association for Research in Vision and Ophthalmology
413
MATERIALS AND METHODS
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
414
mental committee, and in the author's opinion, methods for securing human tissue were humane, included proper consent and approval, and complied with the tenets of the Declaration of Helsinki. Briefly, after washing with Ca2+ and Mg2+ free phosphate buffered saline (PBS), the anterior segment and the vitreous were removed and the sensory retina was discarded. The remaining eye cup was cut into quadrants. Sheets of RPE cells were peeled off from the choroid, recovered by brief centrifugation (800 rpm), and transferred to a 60 mm culture dish containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin G (100 IU/ml), streptomycin (100 jug/ml), and amphotericin B (0.25 mg/ml). RPE cells were cultured in an incubator with a humidified atmosphere of 5% CO2 and 95% air at 37°C. When RPE cells were cultured to near confluency, the cells were washed with PBS solution, incubated for 10 min at 37°C with Ca2+ and Mg2+ free Hank's solution containing 0.02% ethylenediaminetetraacetic acid
(EDTA) and 0.25% trypsin, and subcultured into new culture dishes with a ratio of 1:3. Three to four days after subculture, at the stage of near confluency, RPE cells were harvested. Cells from the third to sixth passages were used in the experiments. In this culture condition, RPE cells were not fully pigmented. About 60 fig of total RNA or 1.5 ^g of polyA+, RNA was prepared from one 100 mm culture dish.
Polymerase Chain Reaction PolyA+ RNA was prepared from the cultured human RPE cells by using Micro-Fast Track (Invitrogen, San Diego, CA). First strand complementary DNA (cDNA) was prepared from the polyA+ RNA using the FirstStrand cDNA synthesis kit (Pharmacia-LKB, Uppsala, Sweden). The primers were synthesized by an oligonucleotide synthesizer (Model 392; Applied Biophysics, Foster City, CA). We chose two highly conserved amino acid sequences by comparing various TGF-/3 sequences and used these to make degenerate primers
l. Amino Acid Sequence Similarity Between the Human TGF-tfl, (32 and #3 Precursors
TABLE
0] 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03
MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRK MH-YCVLSAFLILHLVTVALSLSTCSTLDMDQFMRK MHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKK RIEAIRGQILSKLRLASPP SQGEVPPGPLPEAVLALYNSTR RIEAIRGQILSKLKLTSPPEDYPEPEEVPP EVISIYNSTR RVEAIRGQILSKLRLTSPPEPTVMTH-yP YQVLALYNSTR D R-VAGESAEPEPEPEADYYAKEVTRVLMV---ETH DLLQE-KASRRAAACERERSDE EYYAKEVYKIDMPPFFPSE ELLEEMHGERE-EGC-TQENTES---EYYAKEIHKFDMIQGLAEH NEIYDKFKQSTHSIYMFF NTSELREAVPEPVLLSRAELR NAIPPTFYRPYFRIVRFDVSAMEKNAS NLVKAEFR NELAVCPKGITSKVFRF NVSSVEKNRT---NLFRAEFR LLRL-KLK---VEQHVELYQKYSNNSW RYLSNRLLAPSDS VFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAE VLRVPNPSSKRNEQRIELFQILRPDEHIAK-QRYIGGKNLPTRGT PEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSC GEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIP AEWLSFpyTDTVREWLLRRESNLGLEISIHCPCHTFQP-N-GDIL --DSRDNTLQVDINGFTTGR---RGDIATIHGM -NRPFLL NKSEELEARFAGIDGTSTYTS---GDQKTIKSTRKKNSGKTPHLL --ENIHEVMEIKFKGVDNEDDHFRGDLGRLKK--QK-DHHNPHLI
01 02 03 01 02 03 01 02 03
LMATPLERAQH--LQSS-RHRRALDTNYCFSSTEKNCCVRQLYID LMLLPSYRLES--QQTNRRKKRALDAAYCFRNVQDNCCLRPLYID LMMIPPHRLDNPGQGGQ-RKKRALDTNYCFRNLEENCCVRPLYID FRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQH FKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTI FRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTyLGLYNTL NPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS NPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS NPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS
I "
Dashes are introduced for alignment. Identical amino acids are marked by dots. Highly conserved amino acid sequences are underlined. The arrow indicates the start of the mature TGF-/8 sequences.
TGF-/? Expression in RPE Cells
415
that contained all the possible nucleotide sequences (Tables 1 and 2). Specific primers for TGF-/31, 2, and 3, and TGF-/31 binding protein were synthesized by choosing specific nucleotide sequences from the previously published sequences of human TGF-/31 through 3 10 - 12 and TGF-/31 binding protein13 (Table 2). Primers were designed so that introns were included in expected genomic sequences. Possible amplification of contaminating genomic DNA thus could be detected by the larger size of the polymerase chain reaction (PCR) products. PCR was carried out by the method of Saiki et al,15 with a slight modification.16 The following conditions were used: denaturation, at 94°C for 1.5 min; annealing, 55 or 65°C for 2 min; polymerization, 72°C for 3 min. The reaction was initiated by adding 2 U of Taq polymerase, after which 35 cycles were carried out. Taq DNA polymerase and reagents for PCR experiments were obtained from Perkin-Elmer Cetus (Norwalk, CT). After the reaction, the products were separated by 1.5% agarose gel electrophoresis, and bands of the expected length were extracted. The resulting DNAs were subcloned into the pBluescript II vector (Stratagene, La Jolla, CA). Before subcloning, the vector was treated by Eco RVand the T vectors were made by Taq polymerase and deoxythymidine triphosphate.17 Sequencing of the PCR Products To confirm that PCR products were derived from nucleotide sequences of TGF-/3 isoforms, sequencing of the subcloned DNAs was carried out according to the TABLE 2.
dideoxynucleotide chain termination method18 using a Sequenase ver. 2.0 DNA sequencing kit (United States Biochemicals, Cleveland, OH). Double-strand template DNA was denatured by alkaline treatment, and the sequencing reaction was initiated by adding T3 or T7 primers. Southern and Northern Blot Analysis For Southern blot analysis, 10 /xl of PCR products were separated by 1.5% agarose gel electrophoresis and transferred to a nylon filter (Hybond-N+; Amersham, Buckinghamshire, UK) by the capillary transfer method with 20X standard saline citrate (SSC). The DNA was fixed to the filter by baking at 80°C for 2 hr. DNA fragments corresponding to the target sequences were labeled by an enhanced chemoluminescence gene detection kit (Amersham). Probes used were a 336 bp DNA fragment for human TGF-j81 and a 384 bp DNA fragment for TGF-/32. After hybridization, the filters were washed with the washing buffer (urea 360 g, sodium dodecyl sulfate 4 g, 20X SSC 25 ml/1) twice at 42°C, then with 2X SSC twice at room temperature. PolyA+ RNA preparations were electrophoresed in a 0.8% agarose gel under denaturing conditions and transferred onto a nylon filter using the capillary method. Northern blot analysis was performed according to the method of Thomas.19 Probes used were a 2.1 kbp pair DNA fragment for TGF-/51 that encodes the complete coding and partial flanking sequence of TGF-/31 and the 384 bp DNA fragment for TGF-/32. DNA fragments were labeled with deoxycytidine 5'«-
Primers for Polymerase Chain Reaction
(J) Amino acid and corresponding nucleotide sequences for degenerate primers. TGF-jSs (mixed oligonucleotides) sense anti-sense
(EWLSFD) 5' (DLGWKW) 5'
CAXTGGYTZTCZTTYGA CAYTTCCAZCCZAXXTC
(2) Nucleotide sequences for specific primers. TGF-/31 (expected length, 336 bp) sense anti-sense TGF-/32 (384 bp) sense anti-sense TGF-/33 (300 bp) sense anti-sense TGF-/3 binding protein (467 bp) sense anti-sense
5' 5'
GCCCTGGACACCAACTATTGC GCTGCACTTGCAGGAGCGCAC
5' 5'
TACAGACCCTACTTCAG AAATCTTGCTTCTAGTT
5' 5'
GCACTTGCAAAGGGCTC TTGGCATAGTATTCCGA
5' 5'
TGAGTAACCACACTGGC AACCTGATGTATCTGGA
Amino acid sequences were chosen by comparing human TGF-/31 to 3. 10 " 12 The nucleolide sequence is as follows: A, deoxyadenosine; C, deoxyguanosine; C, deoxycylidine; T, deoxythymidine; X, either A or G; Y, either C or T; Z, either A, G, C, or T.
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
416 32
P triphosphate (Amersham Japan, Tokyo, Japan) by Multiprime DNA labeling system (Amersham). Hybridization was carried out at 42DC in the hybridization buffer containing 50% fonnamide. The final wash was with 0.1X SSC that contained 0.5% sodium dodecyl sulfate at 60°C for 20 min. RESULTS
B 1
2
3
4
1 2
3
4
4
Identification of TGF-/? Isoforms by PCR Using Degenerate Primers PCR using degenerate primers was applied to isolate cDNAs that correspond to the known TGF-jS isoforms or possible new isoforms expressed in human RPE cells. To prepare degenerate primers longer than 17 or 18 nucleotides, five highly conserved amino acid sequences were selected by comparing various human TGF-jS isoforms: EAIRGQILSKL, EWLSFD, DLGWKW, HEPKGY, and EQLSNM (Table 1). Among these sequences, two pairs (EVVLSFD and DLGWKW; HEPKGY and EQLSNM) were chosen for the PCR experiments. Figure. 1 indicates that the products of the expected length were detected from PCR experiments using degenerate primers, (EWLSFD and DLGWKW). The resulting PCR products of the expected length were subctoned into the pBltiescript II vector. Eighteen clones from the PCR experiments were sequenced. Thirteen (72%) out of
-310
FICURE l. Reaction products of PCR experiments using degenerate primers for TGF-/J.
FIGURE 2. Southern blot analysis of PCR products. PCR product (10 M') was separated by agarose gel eteci rophoresis and transferred onto a nitrocellulose filter. The filter was hybridized with the DNA fragment labeled by enhanced chemoluminescence. Labeled probe A, TGF-/32; B, TCF-/3.1. Lane 1, products from PCR using degenerate primers. Lane 2, PCR products for TGF-0 binding proiein. Lane 3, PCR products for TGF-03. Lane 4, PCR products for TGF-02. Lane 5, PCR products for TGF-jSl.
the 18 clones were found to encode nucleotide sequences of human TGF-/32. The remaining five clones (28%) had no amino acid sequences homologous to the known TGF-/3s. These results suggest that human RPE cells primarily express genes of TGF-/32. By Southern blot analysis, one positive band was detected from the PCR products using labeled TGF-/32 DNA fragments as a probe (Fig. 2). Subcloning and subsequent sequencing of the PCR products using another pair of primers (HEPKGY and EQLSNM) revealed that most of the products were derived from other than human TGF-jSs. Identification of TGF-/3 Isoforms by PCR Using Specific Primers To confirm the expression of TGF-/31, 2, and 3, we performed additional PCR experiments using primers encoding the specific nucleotide sequences of TGF/31, 2, and 3. Figure 3 indicates that the PCR products of the expected length were detected for TGF-/31 and 2, but not for TGF-/23, although very faint bands appeared. We carried out one more PCR experiment using the PCR products as the template and tried to discover possible expression of TGF-/33. However, we could not efficiently amplify PCR products of the appropriate length by additional PCR experiments and failed to subclone the products. Thus, expression of TGF-j83 gene was not confirmed by our analysis. We isolated and subcloned PCR products of TGF-/31 and 2 into the pBluescript II vector and sequenced. Again, however, all nucleotide sequences of subcloned PCR products from this experiment revealed sequences
417
TGF-0 Expression in RPE Cells
1 2
3
PCR products, subcloning and subsequent sequencing were performed. Sequencing of the subcloned PCR products revealed that the amplified sequences were derived from TGF-jS binding protein. This indicates that human RPE cells express messages not only of TGF-jS, but also of the TGF-,3 binding protein.
DISCUSSION
- 603 bp -310
FIGURE 3. Reaction products from PCR experiments using specific primers. Lane 1, PCR products for TGF-/33. Lane 2, PCR products for TGF-02. Lane 3, PCR products for TGF-01.
TGF-jS is a cytokine that has been implicated in a number of ocular diseases. For example, a higher concentration of TGF-/? in the vitreous aspirates from eyes with proliferative vitreoretinopathy than from those with nonproliferative retinal detachment has been reported, suggesting important roles of TGF-0 in the process of intraocular proliferation of RPE and other types of the cells.20 TGF-jS induced modification of RPE cell behaviors to growth factors and RPE-mediated gel contraction also have been reported,2'122 although a controversial report also was published.23 This cytokine is known to be widely distributed in the ocular tissues. Also reported has been the recognition of cell growth modulators by anti-TGF-/? antibodies in the aqueous humor and vitreous.20'24'25 Ciliary body epithelial cells embryologically related to the RPE reportedly produce TGF-/31 and 02.26 Also, TGF-/3 im-
A
B
that corresponded to TGF-/31 and 2. Southern blot analysis confirmed that this band was derived from TGF-0] and 2 (Fig. 2). Expression of TGF-jff mRNAs The expression of mRNAs for the TGF-/31 and 2 in the RPE cells was examined by Northern blot analysis. The message for TGF-/31 was detected and the size was 2.5 kbp. The message for TGF-/31 detected by the Northern blot analysis was a single band even when we used DNA fragments that encode the complete coding sequence of TGF-/3! (Fig. 4A). Messages for TGF-02 also were detected, and their sizes were about 6.5 (main band), 5.0, and 4.0 kbp (Figs. 4B, C). PCR and Subsequent Sequencing for Detecting TGF-jS Binding Protein To identify the expression of TGF-/3 binding protein mRNA, we performed PCR experiments using human RPE cDNA as the template and one pair of specific primers that encodes the molecular structure of TGF/31 binding protein.13 PCR experiments showed that the resulting product contained DNA fragments of about 470 bp in size (Fig. 5). To characterize these
kb
I
-9.49 ^7.46 -4.40 -2.37 — 135
FIGURE 4. Northern blot analysis. PolyA+ RNA of 1 jug was elect rophoresed in 0.8% agarose gel under denaturing conditions and transferred onto a nylon filler using capillary method. The final wash was with 0. IX SSC containing- 0.5% sodium dodecyl sulfale at 60°C for 20 min. Lane A, TGF-/J1. Lane B, TGF-/32, overnight exposure. Lane C, TGF-/J2, 1 wk exposure.
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
418
-603 bp -310
FIGURE 5. Reaction products of PCR experiments using specific primers for TGF-/3 binding protein.
munoreactivity was found in adult human retinal photoreceptors. 27 Although RPE cells are thought to produce the cytokine that has an autocrine mechanism to TGF-/S and causes complicated regulation of cellular behaviors,20 production or gene expression of TGF-/? by the cell has not yet been unequivocally demonstrated. Thus, the present study was designed to confirm the gene expression of TGF-/? and to identify isoforms of TGF-/3 expressed in human RPE cells in culture. In this study, we first performed PCR experiments using degenerate primers that encode common ami no acid sequences of TGF-jSl, 2, and 3, because these three isoforms are known to be expressed in mammalian tissues. This technique of PCR is becoming an increasingly popular method for cloning new members of a gene family because of the good possibility that the new members may share common features in the primary structure of the molecules. The TGF-jS superfamily is composed of many members, including TGF-jSl to 5. 28 We thought it was possible that an unknown isoform of TGF-/? was produced in the RPE cells. However, all the subcloned DNA from the PCR experiments using degenerate primers was of TGF-/32 origin. This suggests that the number of copies of cDNA with TGF-/02 origin is much larger than that of other members of the TGF-/? gene family. It is possible that a yet unidentified member of the TGF-/3 gene family is expressed in human RPE cells, but this seems highly unlikely.
To determine the expression of the known members of the TGF-jC? gene family, we performed further PCR experiments using specific primers, because even a relatively small number of cDNA copies can be amplified by this technique. In fact, the PCR experiments showed that TGF-01 also is expressed in RPE cells, but experiments that searched for TGF-/33 were negative. Subsequent sequencing of the subcloned PCR products and Southern blot analysis confirmed that the PCR products were of TGF-/31 origin. Previous studies using bioassay to detect TGF-/3 activity in intraocular fluids such as aqueous humor and vitreous aspirates20-24-25 revealed that most of the immunoreactive TGF-/3 is TGF-/32 and only a small portion is TGFj81. Our data on the preponderance of TGF-/32 gene expression are consistent with these bioassay experiments. Northern blot analysis demonstrated multiple bands for TGF-02. Multiple bands of 6.5, 5.1, and 4.1 kbp for TGF-/32 have been reported in human cell lines, and our data agree with previous reports. 10 ' 29 There are two possible explanations for multiple messages: (1) occurrence of alternative mRNA splicing;29'30 and (2) presence of a highly homologous molecule. The former hypothesis is more likely, because our PCR experiments using degenerate primers did not suggest the presence of highly homologous molecules. Most TGF-/3 is secreted as inactive complexes of high molecular weight.13 Activation of latent TGF-/5 is an important in vivo regulatory step, although little has been elucidated about this mechanism. We have shown that human RPE cells also express the TGF-01 binding protein gene that is thought to be expressed in TGF-/?-producing cells. In summary, we have demonstrated the expression of TGF-/31, TGF-02, and TGF-/3 binding protein genes in RPE cells. Further investigations on modulators produced by healthy or deteriorating RPE cells are needed to understand the pathogenesis of many ocular disorders. Key Words Northern blot analysis, polymerase chain reaction, retinal pigment epithelial cells, transforming growth factor-/?. References 1. Schweigerer L, Malerstein B, Neufeld G, Gospodarowicz D. Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells. Biochem Biophys Res Commun. 1987; 143:934-940. 2. Sternfeld MD, Robertson JE, Sahipley GD, Tsai J, Rosenbaum JT. Cultured human retinal pigment epithelial cells express basic fibroblast growth factors and ks receptors. Curr Eye Res. 1989;8:1 029-1037. 3. Ocrnt A, Fay CT, Parmelee JT. Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment, epithelium. Exp Eye Res. 1991:52:581-589.
TGF-0 Expression in RPE Cells 4. Campochiaro PA, Sugg R, Grotendorst G, Hjelmeland LM. Retinal pigment epithelial cells produce PDGF-like proteins and secrete them into their media. Exp Eye Res. 1989;49:2l7-227. 5. Jaffe GJ, Peters WP, Roberts W, et al. Modulation of macrophage colony stimulating factor in cultured human retinal pigment epithelial cells. Exp Eye Res. 1992; 54:595-603. 6. Elner VM, Scales W, Elner SG, Danfort J, Kunkel SL, Stricter RM. Interleukin-6 (IL-6) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells. Exp Eye Res. 1992;54:361-368. 7. Planck SR, Dang TT, Graves D, Tara D, Ansel JC, Rosenbaum JT. Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1. Invest Ophthalmol Vis Sci. 1992;33:78-82. 8. Elner VM, Strieter RM, Elner AG, Baggiolini M, Lindley I, Kunkel S. Neutrophil chemotactic factor (IL-8) gene expression by cytokine-treated retinal pigment epithelial cells. AmJ Pathol. 1990; 136:745-750. 9. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987; 105:1039-1045. 10. Derynck R, JarrettJA, Chen EY, et al. Human transforming growth factor-/? complementary DNA sequence and expression in normal and transformed cells. Nature. 1985;316:701-705. 11. de Martin R, Haendler B, Hofer-Warbinek R, et al. Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-/3 gene family. EMBO J. 1987;6:3673-3677. 12. Derynck R, Lindquist PB, Lee A, et al. A new type of transforming growth factor-/3, TGF-/33. EMBO J. 1988; 7:3737-3743. 13. Kanzaki T, Olofsson A, Moren A, et al. TGF-01 binding protein: A component of the large latent complex of TGF-/31 with multiple repeat sequences. Cell. 1990:61:1051-1061. 14. Kuriyama S, Ohuchi T, Yoshimura N, Honda Y. Growth factor-induced cytosolic calcium ion transients in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1991;32:2882-2890. 15. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of/3-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230:l 350-1354. 16. Suzuki S, Huang ZS, Tanihara H. Cloning of an integrin j8 subunit exhibiting high homology with integrin 03 subunit. Proc Nad Acad Sci USA. 1990;87:53545358.
419
17. Marchuk D, Drumm M, Saulino A, Collins FS. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991; 19:1154. 18. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977; 74:5463-5467. 19. Thomas PS. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA. 1980; 77:5201-5205. 20. Connor TB, Roberts AB, Sporn MB, et al. Correlation of fibrosis and transforming growth factor-/? type 2 levels in the eye./ Clin Invest. 1989;83:1661-1666. 21. Leschey KH, Hackett SF, Singer JH, Campochiaro PA. Growth factor responsiveness of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1990;31:839-846. 22. Raymond MC, Thompson JT. RPE-mediated collagen gel contraction. Inhibition by colchicine and stimulation by TGF-beta. Invest Ophthalmol Vis Sci. 1990; 31:1079-1086. 23. Opas M, Dziak E. Effects of TGF-beta on retinal pigmented epithelium in vitro. Exp Cell Bio I. 1989; 57:206-212. 24. Eisenstein R, Grant-Bertacchini D. Growth inhibitory activities in avascular tissues are recognized by antitransforming growth factor B antibodies. Curr Eye Res. 1991;10:157-162. 25. Cousins SW, McCabe MM, Danielpour D, Streilein JW. Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest Ophthalmol Vis Sci. 1991; 32:2201-221 1. 26. Helbig H, Kittredge KL, Coca-Prados M, Davis J, Palestine AG, Nussenblatt RB. Mammalian ciliary-body epithelial cells in culture produce transforming growth factor-beta. Graefes Arch Clin Exp Ophthalmol. 1991; 229:84-87. 27. Lutty G, Ikeda K, Chandler C, McLeod DS. Immunohistochemical localization of transforming growth factor-/3 in human photoreceptors. Curr Eye Res. 1991; 10:61-74. 28. Massague J. The transforming growth factor-/? family. Annu Rev Cell Biol. 1990;6:597-641. 29. Webb NR, Madisen L, Rose TM, Purchio AF. Structural and sequence analysis of TGF-02 cDNA clones predicts two different precursor proteins produced by alternative mRNA splicing. DNA. 1988;7:493-497. 30. Kondaiah P, Obberghen-Schilling EV, Ludwig RL, Dhar R, Sporn MB, Roberts AB. cDNA cloning of porcine transforming growth factor-/?! mRNAs. Evidence for alternate splicing and polyadenylation. J Biol Chem. 1988;263:18313-18317.
