The journal of Dermatology Vol. 19: 642-643, 1992
International Workshop I (WS I) The Role of Growth Factors in Wound Healing WS I-I
Connective Tissue Growth Factor Atsuyuki Igarashi, Douglass M. Bradham*, Hitoshi Okochi* and Gary R. Grotendorst* Connective tissue growth factor (CTGF) is secreted by human umbilical vein endothelial cells and has a biological activity related to that of platelet-derived growth factor (PDGF). Human umbilical vein endothelial cells have been previously reported to secrete PDGF-related factors into the media. DiCorleto et at. reported that endothelial cells secrete factors that are chemotactic and mitogenic for connective tissue cells and around 30% of this activity was neutralized by antihuman PDGF antibodies (1). Collins et at. reported that human umbilical vein endothelial cells express both the A and B chain genes of PDGF (2, 3). Thus the mitoattractant activity found in human umbilical vein endothelial cells was thought to be due to PDGF gene products. However, the exact nature of this protein was uncertain. We tried to determine the properties of the PDGF-related molecules secreted by human umbilical vein endothelial cells and found a new peptide distinct from PDGF (4). Confluent human umbilical vein endothelial cells were incubated in serum free media for 6 and 48 hours. Then conditioned media was recovered and analyzed by Western blotting which was probed with anti-PDGF antibody. We detected 36-38kd molecules which were bigger than PDGF B chain dimers. Neither anti amino terminal A chain nor anticarboxy terminal B chain antibody picked up this band, indicating that this peptide does not contain PDGF A or B chain peptides. In reduced conditions, the band appeared at 38kd, indicating that this peptide is a monomer. We termed this protein CTGF. We purified CTGF from the conditioned media using an affinity column made with the anti-PDGF antibody and an Affigel 10 support. We performed chemotactic and mitogenic assays using NIH3T3 cells. These data show that CTGF has biological activities affecting these cells, as do PDGF isoforms. Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo,japan. *Department of Anatomy and Molecular Biology, University of Miami, USA.
CCCGGCCGACAACCCtCiAGo\CGACAGCCtGGCGCGTCCCGG1CCCtACCTCCGAtCACCGtCAGCGCTCCAGGlXttGCGCTccctGCTCGCCGCCACC GCGCCCTCCGCTecccCCGCAGTGCCMCC
ATG Ace ace Gee ACT "T' GGC CCCGTt tee GTt eee TTl: GTGGTe eTC: eTC _~AI."I.~~'~~~~~A~~~~~L~
Get etc Toe AGe CGG eee eee GTC GGC CAG Me TGCAGe GOO CCG fCC eGG fCC ttG GAt
cwo
tCG GCG CCG CGC
~~~~~~M.~~~"_~~_~~~~~_au~~~~
TCCtCG GtG GGC GIG AGe
ere
GrG eTG GACGGC fOC GOt TGC tee cGC GTe TOCeee ANi CAG eTC GGC GAGera
99 110
17 255
u
no
~~AI.M~~~~~~ _ _ ~G~~~~~~AI.~~~'lyG~~ ~
fCC Ace GAG tee GAC etc TOC GAt tao CAe MG GGC etc TTC ret GAt TTC liCe tee ttQ see AAC cat MG ATC
a-.. Pro
405
"s l.,.. Sly l ...... cy. Alp . . . Cly .... Pro At. . . . A,... l.,.. Iia
92
GGC GT' TIX Ace Get AM lOAT GaT GCT CCC TGC ATC fTC GGT GGT ACG GTG TACCOCAGe GGA GAG TCe tte CAG ely '1.1 eys 11lr AI. l.,.. . . . .I., AI. P,. C"r'S lIa .....Iy .. Iy 11lr vel I". . , . 1M' "'y Illy 1M' . . . "In
410 117
AGe AGe TIX AAG TAC CAG TGe ACG TGC eTG GAt GOO GCG GTC GGC TGe ATG eee eTG TOCAGe ATC GAt GTTCGT sa.. c.,.. l.,.. T,.. Cln eys Tttr cy. l . . Alp ely Ala l1li1 GIl' 0;lI * t Pro l . . Cya . . . IIIIt ... wei ArI
555 142
CHi cee AGe cet GAt TGC ceette eCG AGG AGIO GTe MG CTG eec GGG AM TOCTGC GAG GAG T" GTG TGT GAt l . . P,.. ... " . "'" c.,.. Pro ... Pro Ar"I A,... Yal L.... leu Pro lily l .... c.,.. Cys Illy Illy Trp V.I c.,.."'"
6JO
GAG cee MG GAt W ACC GTlO Gn GGG CeT ccc eTC Gee ccr TAC eGA CTG"'" ~c Act TTT CGC CCA GAt eCA Glu Pro l.,.. "'" GIn n.r '1.1 '1.1 Gly Pro At. Leu AI. AI.-Tyr Ani Leu Glu A. n.r "'- G1y Pro "... Pro
705
ey. T1w CI,,",.. _ , , . . c:y.
sar
167
192
ACT ATG An AliAGce ......C IGC eTC GTe CAG Ace ACA &AG TOO AGC Get TGT TCCMG Act TGT GGG ATCi GGC Arc Tttr MH lie,," Ala t.n Cyw. Leu '1.1 Gin Thr Tttr Gly Tilt ... Ala Cys . . . L.... ",r c.,. Illy IIIIt Illy II.
710 217
tee Ace
eGG GTT ACt MT COAt Me ccc tee fGC AGG CfA GAG MG CAG AOC eGC eTG TOCATGGTe AGG ccr fGC sar 11Ir Art Y.I_",r ... Asp.b!:! Ala _ CyIi Ar"I L., ely l.,.. Iln .... A,... l . . c:.,.. MIt Val Art P,.. c.,.
855 242
GAA GCT GAC eTG GAA GAG .u.c An ......G ......G GGC AM MG TGC ATCeGT ACT cee AM ATC TCC Me CCT ATe Mel elu AI. "'" leu IIty elu Aan n. L)'II L,.. Illy Lye L.,. CyIi II.Ar"I Thr 10,.. L)'II II. 1M' l.,. P,.. lie l.,.
930 267
TTTGAGCTTin~tGC~AGCA'GMG~'~~~WtTeTGT~~TetACtGACGGC~TGC1~
,.. Glu leu ... III.,
c.,.. Thr s.r
Met lye Ttlr T,.. A,... Ala l.,.. PM Cya Illy Yal
c.,.. Thr
Up
CI., "., C'rs
29l
~~~~~~ACC~CtGCCG~&AGTTCMGTGC«TGAC~GAGGreAtGMGMG.u.cATGt_
CysTttr'""ls".,Tttr11lr11lr~,",~GIUPMl""Cyw.,",_GIY~~~lyel.,.. ATGTTeATCAAG~tGTGetTGCWTAC.u.cTGT~~GAC
nw Cyw.
Als C'rs .ts T,.,. ... C'rs Pro III.,
... 1IIIt
517
TGACA'eTTTWT~CTGmTAC~'I»
Up II ...... Clu .... l ... T,.. TY" ArI
342
AAG ATG tAC liGA GAt ATG GCA T'" AGCCAGAGAGTGAlOAlOACAn.uctCAlTAGACTGGAACTTGMCTGATTCACATCTCATTnTCC IIIIt ely'" IIIIt AI. T.,..
1245 349
Met ..... II. l.,..
l.,.
T,..
GTAAAAATGAnTCAGTAGCAWGTTWLWTCTGTTTTT,pAACTGGCGGAA.uGATTCCCACCCAATTCAAAACATtGTGCCATGlCAAACAM 1344 TAc;TCTATCTTCCCCAQACACTGGTT1GMCMtctTAAGACTTGACAGTCGAACTACATtACiTACACAGCACCAliAATCiTATATTAACGTGfGGC:TtT 1443 AGGAGCAGTGGGAGQGTACCGGCCCCGlTAGTATCATC4GA1CGACTCTTA1ACGAGt""TATGCCTGCTATTTGAACiTliTMTTGAliAAGGAAAATTT 1542 tAGCG1GClCAC:TGACCTGCCTGTAGCCCtAGTGACAGCTACGATGTCCATTCfCCMCCAfCMGAGAC1GAGrCMGTTGTTCCTTAAGTCAGAAtA ,,,, GCAGACTtAGCTtTGACATTeTGATTeliAATGACACTGTTCAGGAATCGliAATCCfGT,,-,TTlGACTGGACAGCTTGTlOGCMGlGAATTTGCtTGlA 1740 ACAAliCCAGATTTTTTAAM...!!!!TATTCiTWTATTGTCTGTGTGTGTGTGtGTGTATATAfATATATATATGTACAGTTATCTAAGTTA6I!.IAAAG I&)9 TTGTT1GTGCtTTTTJ,I,TTTnCiTTTTTAATGCTTtGATATTTWHiTTAGCCTtAATTTCTGAACACCA1AGQTAGAATGTWGCTTGTCTlOATCG 1931 TTCAAAGtATGAAATGGATACTTAtATGGAMTTCTGCTCAGATAGAATt>AcAGTCCliTCMAAtAGATTGTtTCCAAAGGGGAGGCAtCACTGTCTTC 2037 /OCAGGCTGA1TTC1AGGTAGGA.UTGTGGTAGCTCAeG2075
Fig. l. Nucleotide sequence and amino acid translation of CTGF eDNA from human vascular endothelial cells. The open reading frame for the CTGF protein extends from the ATG initiation site at nucleotide 130 to the TGA site at II 77. The possible glycosylation sites at asparagines 28 and 225 are underlined. The 3' region contains three ATTTA sites which are underlined.
Next we performed a receptor competition assay. CTGF binding to cell surfaces competed with PDGF BB, indicating that CTGF and PDGF share the same receptor. It seemed to be very difficult to determine the amino acid sequence of these proteins, because the amount of CTGF recovered from the conditioned media was very scant. Therefore we screened the human umbilical vein endothelial cell cDNA library made in lambda gtll using the anti-PDGF antibody. Over five hundred thousand recombinant clones were screened, and we picked up several positive ones. Two of these were fragments ofPDGF B chain
Connective Tissue Crowth Factor CTGr {3IG-M2 Cyr-51 CEP-1O
* PCRCPDEPA ** * ** * * * MTAASMG PYRY ArYYLLALC* SRPA YGQNCSG PRC*PAG YSLY LDGCGCCRVCAKQLGELCTERDPCDPHKG -L-SVA--I SL-L ------ T- --T--D- -AQ-Q-- .1.1 - .1--H- - ---- - -- - ------ - - --- ----------- ----SSSTrRTLA-- -T--H- T-L-LS T-PPA-H--L- --K--AP--G--R------K------N-D-SKTQ---HT-MGSAGA-P- LAAA -L - LA- L- L-SP-PA V-Q-- A .1--Q-AP--G--P--- ---K ----- -N-D-SRTQ- - -HT--
CTGr {3IG-M2 Cyr-51 CEP-IO
* DGAPC*I PGGTVYRSGESPQSSCK * YQCTCLDGA ** * * * LrCOrGSPANRK 1GVCTAK YGCMPLCSMDVRLPSPDCPrPRRVKLPG ------------------- -----v-- -S--- - - - - - - - - - -- - - --- -- - -- - V-- - ---- - - - ---- - -- - -- - ---E-N--ASSTALK - I -R-QSE-R--EYNSR I -QN-----PN--H----I-- - --- I ---PQELS--NLG--N--L--VS-E-N- - ASPAATN-I -R-QSE- R-- EYNSK I -QN-- - - - PN- -H- - - -I - - -- - - I - -- PQELS-- NLG- -S- - L- -V--
CTGP {J IG-M2 Cyr-51 CEP-IO
KCCEEWVCDEPKD QTVVGPALAAYRLEDTrGPDPTMI RAN CLVQTTE -- - - -- -- ---- R- A--- - - - -- ------------H- -- -----Q-- - - ----- DS I KDSLDDQDDLLGLDASEVELTRNNEL I AI GKGSSLKRLPV --TE-RVLrNPLHAHGQK- -----S Q---------S--ALEELEGNPSKErGLOASEGELTRNNELI AI VKGG LKHLPV--SE-QSRAPE NPK- I ----S
CTGP {J IG-H2 Cyr-51 CEF-ID
WSACSKTCGHG 1STRVTNONASCRLEKQSRLCHVRPCEADLEEN I KKGKKC I RTPK I SKPI KPELSGCTSHKTYRAKFCG - - - - - - - - -- - - - - - -- ---TF- - - - - - - - - - - - - - - - - - - -- -- - -- - - - - - -- -- A--V- - - - - - -- - v- - -- - -- ---Q- --S--T---------- PE---V-ET- I -E----GQPVYSSL- -----SK-K-SPE-VR-TYA --S-V -K-- P-Y---Q------T----------PD-K- I -ET- I -E----GQPSYASL------TK-K-SPS-VR-TYA--S-V-K--P-Y--
CTGF (J IG-H2 Cyr-51 CEF-IO
*
**
*
**
*
*
**
*
• ** * ** * VCTDGRCCTPHRTTTLPVEFKCPDGEVHKKNHHF I KTCACHYNCPGOND I FESLYYRKHYGDHA
*
- - - - -- - - - - - -- -- - - - - -- - - -- - I - -- - - - - - - -- -- - - - - -- - -- -- - - - - -- - -- - - -S-V-- -- --- LQ-R-VKHR-R-E---MFS--V-H-QS-K-N----HPNEASFR-- SLFN- I HKFRD S-V- ------QQ-R-VK I R-R-D---TFT-SV-H-QS-R- N----HA-EAYP F-- LVNN- I HKFRD
cDNA. The third one was an unknown cDNA; we rescreened the library with this cDNA and got a cDNA clone which contained a 2,100 base pair insert. We considered this clone to be CTGF cDNA, subcloned it into MI3 phage vectors, and completed a nucleotide sequence. Figure I shows the nucleotide sequence and amino acid translation ofCTGF cDNA from human umbilical vein endothelial cells. This has an open reading frame of 1,047 base pairs encoding 349 amino acids, which correspond to a 38kd molecular weight protein. The amino terminal of the peptide contains a .hydrophobic signal sequence indicating that CTCF is a secreted protein. This chain also contains 39 cysteine residues, indicating that this peptide has a complex structure with multiple intra molecular disulfide bonds. There is a 40% sequence homology between the CTGF cDNA and the cDNA for both the A and B chains of PDGF. Next we compared the CTGF mRNA expression in human umbilical vein endothelial cells, human foreskin fibroblasts, retinal pigmented epithelial cells, neutrophils, and monocytes. We detected the 2.4kb of CTGF transcript in human foreskin fibroblasts, especially when treated with TGF beta, and in retinal pigmented epithelial cells. Neither neutrophils nor monocytes showed a CTGF transcript Recently, several gene products similar to CTGF have been reported. Fig. 2 shows the amino acid sequence comparison of these proteins. Beta IG-M2 is induced by TGF beta in mouse embryo fibroblasts, and it has a 90% homology to CTGF. The function of beta IG-M2 in vivo is unknown, but we assume that this peptide represents the CTGF of mice and that CTGF has a low species-specificity. From these results, we suggest that the CTGF gene is phylogenically well conserved and has some imPOrtant biological role.
*
64~
79 78 75 75 158 157 155 155
205 204 233
230 285
284 313·
310
349
348 379 375
Fig. 2. Comparison of amino acid sequences of the translated cDNA for CTGF, beta IG-M2, CYR61, and CEF-IO. Beta IG-M2 has a 90% homology to CTGF. CTGF has a 45% homology to CYR61 and CEF-IO.
CEF-IO is a product of chicken embryo fibroblasts. Cyr61 is found in mouse BALB/c 3T3 fibroblasts. These products are cysteine rich, and all the cysteine residues are conserved with few gaps in the sequence. CTGF is a novel growth factor which shows a PDGF-like activity. The regulation of CTGF gene expression seems to be distinct from other known growth factor regulated genes. TGF beta is known to have various biological effects on cell proliferation. Recently TGF beta has been suspected as the cause of fibrotic disorders (5), in which CTGF may act as a mediator in abnormal cell proliferation. The data presented here propose that CTGF may play an important role in the growth regulation of connective tissue during wound healing, tissue regeneration, and in the pathogenesis of connective tissue disorders.
References I) DiCorleto PE: Cultured endothelial cells produce multiple growth factors for connective tissue cells, Exp Cell Res, 153: 167-172, 1984. 2) Collins T, Ginsburg D, Boss JM, Orkin SH, PoberJS: Cultured human endothelial cells express plateletderived growth factor B chain: cDNA cloning and structural analysis, Nature (Lond), 316: 748-750, 1985. 3) Collins T, Bonthron DT, Orkin SH: Alternative splicing affects function of encoded platelet-derived growth factor A chain, Nature (Lond), 328: 621-624, 1987. 4) Bradham DM, Igarashi A, Potter RL, Grotendorst GR: Connective tissue growth factor: A cysteine-rich mitogen secreted by human vascular endothelial cell is related to the SRC-induced immediate early gene product CEF-lO,J Cell Biol, 114: 1285-1294, 1991. 5) Smith EA, LeRoy EC: A possible role for transforming growth factor beta in systemic sclerosis, ] Invest Dermatol, 95: 125S-127S.
International Workshop I (WS I) The Role of Growth Factors in Wound Healing WS I-I
Connective Tissue Growth Factor Atsuyuki Igarashi, Douglass M. Bradham*, Hitoshi Okochi* and Gary R. Grotendorst* Connective tissue growth factor (CTGF) is secreted by human umbilical vein endothelial cells and has a biological activity related to that of platelet-derived growth factor (PDGF). Human umbilical vein endothelial cells have been previously reported to secrete PDGF-related factors into the media. DiCorleto et at. reported that endothelial cells secrete factors that are chemotactic and mitogenic for connective tissue cells and around 30% of this activity was neutralized by antihuman PDGF antibodies (1). Collins et at. reported that human umbilical vein endothelial cells express both the A and B chain genes of PDGF (2, 3). Thus the mitoattractant activity found in human umbilical vein endothelial cells was thought to be due to PDGF gene products. However, the exact nature of this protein was uncertain. We tried to determine the properties of the PDGF-related molecules secreted by human umbilical vein endothelial cells and found a new peptide distinct from PDGF (4). Confluent human umbilical vein endothelial cells were incubated in serum free media for 6 and 48 hours. Then conditioned media was recovered and analyzed by Western blotting which was probed with anti-PDGF antibody. We detected 36-38kd molecules which were bigger than PDGF B chain dimers. Neither anti amino terminal A chain nor anticarboxy terminal B chain antibody picked up this band, indicating that this peptide does not contain PDGF A or B chain peptides. In reduced conditions, the band appeared at 38kd, indicating that this peptide is a monomer. We termed this protein CTGF. We purified CTGF from the conditioned media using an affinity column made with the anti-PDGF antibody and an Affigel 10 support. We performed chemotactic and mitogenic assays using NIH3T3 cells. These data show that CTGF has biological activities affecting these cells, as do PDGF isoforms. Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo,japan. *Department of Anatomy and Molecular Biology, University of Miami, USA.
CCCGGCCGACAACCCtCiAGo\CGACAGCCtGGCGCGTCCCGG1CCCtACCTCCGAtCACCGtCAGCGCTCCAGGlXttGCGCTccctGCTCGCCGCCACC GCGCCCTCCGCTecccCCGCAGTGCCMCC
ATG Ace ace Gee ACT "T' GGC CCCGTt tee GTt eee TTl: GTGGTe eTC: eTC _~AI."I.~~'~~~~~A~~~~~L~
Get etc Toe AGe CGG eee eee GTC GGC CAG Me TGCAGe GOO CCG fCC eGG fCC ttG GAt
cwo
tCG GCG CCG CGC
~~~~~~M.~~~"_~~_~~~~~_au~~~~
TCCtCG GtG GGC GIG AGe
ere
GrG eTG GACGGC fOC GOt TGC tee cGC GTe TOCeee ANi CAG eTC GGC GAGera
99 110
17 255
u
no
~~AI.M~~~~~~ _ _ ~G~~~~~~AI.~~~'lyG~~ ~
fCC Ace GAG tee GAC etc TOC GAt tao CAe MG GGC etc TTC ret GAt TTC liCe tee ttQ see AAC cat MG ATC
a-.. Pro
405
"s l.,.. Sly l ...... cy. Alp . . . Cly .... Pro At. . . . A,... l.,.. Iia
92
GGC GT' TIX Ace Get AM lOAT GaT GCT CCC TGC ATC fTC GGT GGT ACG GTG TACCOCAGe GGA GAG TCe tte CAG ely '1.1 eys 11lr AI. l.,.. . . . .I., AI. P,. C"r'S lIa .....Iy .. Iy 11lr vel I". . , . 1M' "'y Illy 1M' . . . "In
410 117
AGe AGe TIX AAG TAC CAG TGe ACG TGC eTG GAt GOO GCG GTC GGC TGe ATG eee eTG TOCAGe ATC GAt GTTCGT sa.. c.,.. l.,.. T,.. Cln eys Tttr cy. l . . Alp ely Ala l1li1 GIl' 0;lI * t Pro l . . Cya . . . IIIIt ... wei ArI
555 142
CHi cee AGe cet GAt TGC ceette eCG AGG AGIO GTe MG CTG eec GGG AM TOCTGC GAG GAG T" GTG TGT GAt l . . P,.. ... " . "'" c.,.. Pro ... Pro Ar"I A,... Yal L.... leu Pro lily l .... c.,.. Cys Illy Illy Trp V.I c.,.."'"
6JO
GAG cee MG GAt W ACC GTlO Gn GGG CeT ccc eTC Gee ccr TAC eGA CTG"'" ~c Act TTT CGC CCA GAt eCA Glu Pro l.,.. "'" GIn n.r '1.1 '1.1 Gly Pro At. Leu AI. AI.-Tyr Ani Leu Glu A. n.r "'- G1y Pro "... Pro
705
ey. T1w CI,,",.. _ , , . . c:y.
sar
167
192
ACT ATG An AliAGce ......C IGC eTC GTe CAG Ace ACA &AG TOO AGC Get TGT TCCMG Act TGT GGG ATCi GGC Arc Tttr MH lie,," Ala t.n Cyw. Leu '1.1 Gin Thr Tttr Gly Tilt ... Ala Cys . . . L.... ",r c.,. Illy IIIIt Illy II.
710 217
tee Ace
eGG GTT ACt MT COAt Me ccc tee fGC AGG CfA GAG MG CAG AOC eGC eTG TOCATGGTe AGG ccr fGC sar 11Ir Art Y.I_",r ... Asp.b!:! Ala _ CyIi Ar"I L., ely l.,.. Iln .... A,... l . . c:.,.. MIt Val Art P,.. c.,.
855 242
GAA GCT GAC eTG GAA GAG .u.c An ......G ......G GGC AM MG TGC ATCeGT ACT cee AM ATC TCC Me CCT ATe Mel elu AI. "'" leu IIty elu Aan n. L)'II L,.. Illy Lye L.,. CyIi II.Ar"I Thr 10,.. L)'II II. 1M' l.,. P,.. lie l.,.
930 267
TTTGAGCTTin~tGC~AGCA'GMG~'~~~WtTeTGT~~TetACtGACGGC~TGC1~
,.. Glu leu ... III.,
c.,.. Thr s.r
Met lye Ttlr T,.. A,... Ala l.,.. PM Cya Illy Yal
c.,.. Thr
Up
CI., "., C'rs
29l
~~~~~~ACC~CtGCCG~&AGTTCMGTGC«TGAC~GAGGreAtGMGMG.u.cATGt_
CysTttr'""ls".,Tttr11lr11lr~,",~GIUPMl""Cyw.,",_GIY~~~lyel.,.. ATGTTeATCAAG~tGTGetTGCWTAC.u.cTGT~~GAC
nw Cyw.
Als C'rs .ts T,.,. ... C'rs Pro III.,
... 1IIIt
517
TGACA'eTTTWT~CTGmTAC~'I»
Up II ...... Clu .... l ... T,.. TY" ArI
342
AAG ATG tAC liGA GAt ATG GCA T'" AGCCAGAGAGTGAlOAlOACAn.uctCAlTAGACTGGAACTTGMCTGATTCACATCTCATTnTCC IIIIt ely'" IIIIt AI. T.,..
1245 349
Met ..... II. l.,..
l.,.
T,..
GTAAAAATGAnTCAGTAGCAWGTTWLWTCTGTTTTT,pAACTGGCGGAA.uGATTCCCACCCAATTCAAAACATtGTGCCATGlCAAACAM 1344 TAc;TCTATCTTCCCCAQACACTGGTT1GMCMtctTAAGACTTGACAGTCGAACTACATtACiTACACAGCACCAliAATCiTATATTAACGTGfGGC:TtT 1443 AGGAGCAGTGGGAGQGTACCGGCCCCGlTAGTATCATC4GA1CGACTCTTA1ACGAGt""TATGCCTGCTATTTGAACiTliTMTTGAliAAGGAAAATTT 1542 tAGCG1GClCAC:TGACCTGCCTGTAGCCCtAGTGACAGCTACGATGTCCATTCfCCMCCAfCMGAGAC1GAGrCMGTTGTTCCTTAAGTCAGAAtA ,,,, GCAGACTtAGCTtTGACATTeTGATTeliAATGACACTGTTCAGGAATCGliAATCCfGT,,-,TTlGACTGGACAGCTTGTlOGCMGlGAATTTGCtTGlA 1740 ACAAliCCAGATTTTTTAAM...!!!!TATTCiTWTATTGTCTGTGTGTGTGTGtGTGTATATAfATATATATATGTACAGTTATCTAAGTTA6I!.IAAAG I&)9 TTGTT1GTGCtTTTTJ,I,TTTnCiTTTTTAATGCTTtGATATTTWHiTTAGCCTtAATTTCTGAACACCA1AGQTAGAATGTWGCTTGTCTlOATCG 1931 TTCAAAGtATGAAATGGATACTTAtATGGAMTTCTGCTCAGATAGAATt>AcAGTCCliTCMAAtAGATTGTtTCCAAAGGGGAGGCAtCACTGTCTTC 2037 /OCAGGCTGA1TTC1AGGTAGGA.UTGTGGTAGCTCAeG2075
Fig. l. Nucleotide sequence and amino acid translation of CTGF eDNA from human vascular endothelial cells. The open reading frame for the CTGF protein extends from the ATG initiation site at nucleotide 130 to the TGA site at II 77. The possible glycosylation sites at asparagines 28 and 225 are underlined. The 3' region contains three ATTTA sites which are underlined.
Next we performed a receptor competition assay. CTGF binding to cell surfaces competed with PDGF BB, indicating that CTGF and PDGF share the same receptor. It seemed to be very difficult to determine the amino acid sequence of these proteins, because the amount of CTGF recovered from the conditioned media was very scant. Therefore we screened the human umbilical vein endothelial cell cDNA library made in lambda gtll using the anti-PDGF antibody. Over five hundred thousand recombinant clones were screened, and we picked up several positive ones. Two of these were fragments ofPDGF B chain
Connective Tissue Crowth Factor CTGr {3IG-M2 Cyr-51 CEP-1O
* PCRCPDEPA ** * ** * * * MTAASMG PYRY ArYYLLALC* SRPA YGQNCSG PRC*PAG YSLY LDGCGCCRVCAKQLGELCTERDPCDPHKG -L-SVA--I SL-L ------ T- --T--D- -AQ-Q-- .1.1 - .1--H- - ---- - -- - ------ - - --- ----------- ----SSSTrRTLA-- -T--H- T-L-LS T-PPA-H--L- --K--AP--G--R------K------N-D-SKTQ---HT-MGSAGA-P- LAAA -L - LA- L- L-SP-PA V-Q-- A .1--Q-AP--G--P--- ---K ----- -N-D-SRTQ- - -HT--
CTGr {3IG-M2 Cyr-51 CEP-IO
* DGAPC*I PGGTVYRSGESPQSSCK * YQCTCLDGA ** * * * LrCOrGSPANRK 1GVCTAK YGCMPLCSMDVRLPSPDCPrPRRVKLPG ------------------- -----v-- -S--- - - - - - - - - - -- - - --- -- - -- - V-- - ---- - - - ---- - -- - -- - ---E-N--ASSTALK - I -R-QSE-R--EYNSR I -QN-----PN--H----I-- - --- I ---PQELS--NLG--N--L--VS-E-N- - ASPAATN-I -R-QSE- R-- EYNSK I -QN-- - - - PN- -H- - - -I - - -- - - I - -- PQELS-- NLG- -S- - L- -V--
CTGP {J IG-M2 Cyr-51 CEP-IO
KCCEEWVCDEPKD QTVVGPALAAYRLEDTrGPDPTMI RAN CLVQTTE -- - - -- -- ---- R- A--- - - - -- ------------H- -- -----Q-- - - ----- DS I KDSLDDQDDLLGLDASEVELTRNNEL I AI GKGSSLKRLPV --TE-RVLrNPLHAHGQK- -----S Q---------S--ALEELEGNPSKErGLOASEGELTRNNELI AI VKGG LKHLPV--SE-QSRAPE NPK- I ----S
CTGP {J IG-H2 Cyr-51 CEF-ID
WSACSKTCGHG 1STRVTNONASCRLEKQSRLCHVRPCEADLEEN I KKGKKC I RTPK I SKPI KPELSGCTSHKTYRAKFCG - - - - - - - - -- - - - - - -- ---TF- - - - - - - - - - - - - - - - - - - -- -- - -- - - - - - -- -- A--V- - - - - - -- - v- - -- - -- ---Q- --S--T---------- PE---V-ET- I -E----GQPVYSSL- -----SK-K-SPE-VR-TYA --S-V -K-- P-Y---Q------T----------PD-K- I -ET- I -E----GQPSYASL------TK-K-SPS-VR-TYA--S-V-K--P-Y--
CTGF (J IG-H2 Cyr-51 CEF-IO
*
**
*
**
*
*
**
*
• ** * ** * VCTDGRCCTPHRTTTLPVEFKCPDGEVHKKNHHF I KTCACHYNCPGOND I FESLYYRKHYGDHA
*
- - - - -- - - - - - -- -- - - - - -- - - -- - I - -- - - - - - - -- -- - - - - -- - -- -- - - - - -- - -- - - -S-V-- -- --- LQ-R-VKHR-R-E---MFS--V-H-QS-K-N----HPNEASFR-- SLFN- I HKFRD S-V- ------QQ-R-VK I R-R-D---TFT-SV-H-QS-R- N----HA-EAYP F-- LVNN- I HKFRD
cDNA. The third one was an unknown cDNA; we rescreened the library with this cDNA and got a cDNA clone which contained a 2,100 base pair insert. We considered this clone to be CTGF cDNA, subcloned it into MI3 phage vectors, and completed a nucleotide sequence. Figure I shows the nucleotide sequence and amino acid translation ofCTGF cDNA from human umbilical vein endothelial cells. This has an open reading frame of 1,047 base pairs encoding 349 amino acids, which correspond to a 38kd molecular weight protein. The amino terminal of the peptide contains a .hydrophobic signal sequence indicating that CTCF is a secreted protein. This chain also contains 39 cysteine residues, indicating that this peptide has a complex structure with multiple intra molecular disulfide bonds. There is a 40% sequence homology between the CTGF cDNA and the cDNA for both the A and B chains of PDGF. Next we compared the CTGF mRNA expression in human umbilical vein endothelial cells, human foreskin fibroblasts, retinal pigmented epithelial cells, neutrophils, and monocytes. We detected the 2.4kb of CTGF transcript in human foreskin fibroblasts, especially when treated with TGF beta, and in retinal pigmented epithelial cells. Neither neutrophils nor monocytes showed a CTGF transcript Recently, several gene products similar to CTGF have been reported. Fig. 2 shows the amino acid sequence comparison of these proteins. Beta IG-M2 is induced by TGF beta in mouse embryo fibroblasts, and it has a 90% homology to CTGF. The function of beta IG-M2 in vivo is unknown, but we assume that this peptide represents the CTGF of mice and that CTGF has a low species-specificity. From these results, we suggest that the CTGF gene is phylogenically well conserved and has some imPOrtant biological role.
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64~
79 78 75 75 158 157 155 155
205 204 233
230 285
284 313·
310
349
348 379 375
Fig. 2. Comparison of amino acid sequences of the translated cDNA for CTGF, beta IG-M2, CYR61, and CEF-IO. Beta IG-M2 has a 90% homology to CTGF. CTGF has a 45% homology to CYR61 and CEF-IO.
CEF-IO is a product of chicken embryo fibroblasts. Cyr61 is found in mouse BALB/c 3T3 fibroblasts. These products are cysteine rich, and all the cysteine residues are conserved with few gaps in the sequence. CTGF is a novel growth factor which shows a PDGF-like activity. The regulation of CTGF gene expression seems to be distinct from other known growth factor regulated genes. TGF beta is known to have various biological effects on cell proliferation. Recently TGF beta has been suspected as the cause of fibrotic disorders (5), in which CTGF may act as a mediator in abnormal cell proliferation. The data presented here propose that CTGF may play an important role in the growth regulation of connective tissue during wound healing, tissue regeneration, and in the pathogenesis of connective tissue disorders.
References I) DiCorleto PE: Cultured endothelial cells produce multiple growth factors for connective tissue cells, Exp Cell Res, 153: 167-172, 1984. 2) Collins T, Ginsburg D, Boss JM, Orkin SH, PoberJS: Cultured human endothelial cells express plateletderived growth factor B chain: cDNA cloning and structural analysis, Nature (Lond), 316: 748-750, 1985. 3) Collins T, Bonthron DT, Orkin SH: Alternative splicing affects function of encoded platelet-derived growth factor A chain, Nature (Lond), 328: 621-624, 1987. 4) Bradham DM, Igarashi A, Potter RL, Grotendorst GR: Connective tissue growth factor: A cysteine-rich mitogen secreted by human vascular endothelial cell is related to the SRC-induced immediate early gene product CEF-lO,J Cell Biol, 114: 1285-1294, 1991. 5) Smith EA, LeRoy EC: A possible role for transforming growth factor beta in systemic sclerosis, ] Invest Dermatol, 95: 125S-127S.
