Hammerhead ribozyme targeting connective tissue growth factor mRNA blocks transforming growth factor-beta mediated cell proliferation.
OBJECTIVE
Excessive scarring following trauma or surgery of cornea, conjunctiva or retina can greatly impair visual outcome. At present, no agents are clinically available that selectively reduce activity of genes that regulate fibrosis. Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues, including cornea and conjunctiva. In this study, hammerhead ribozymes targeting CTGF mRNA were synthesized, kinetic parameters were measured, and the effect on TGF-beta-mediated cell proliferation was measured in cultured human fibroblasts.


METHODS
The mRNA sequence of human CTGF was scanned for potential hammerhead ribozyme cleavage sites, and predicted secondary folding structures around the sites were calculated. Synthetic 12mer ribozymes and 33mer oligonucleotide mRNA targets corresponding to two sites were synthesized, and kinetic constants calculated from Hanes-Wolff plots of in vitro cleavage reactions. The ribozyme with higher percentage cleavage and kinetic rate was cloned into an expression plasmid (pTR-UF21) and stably transfected into cultured human fibroblasts. An inactive ribozyme plasmid served as a negative control. The effects of the ribozyme on expression of TGF-beta-induced CTGF mRNA and protein levels were measured using ELISA and real-time TaqMan quantitative RT-PCR. Finally, the effect of the CTGF ribozyme on TGF-beta-mediated proliferation of fibroblasts was measured using a non-radioactive cell proliferation microtiter assay.


RESULTS
Of the eight potential hammerhead ribozyme cleavage sites in human CTGF mRNA, two sites (CHR 745, and CHR 859) were identified with optimal secondary folding. CHR 859 cleaved 94% of the target mRNA, compared to 46% cleavage for CHR 745 after 16 hr of reaction. CHR 859 had a K(m) of 1.56 microM and a K(cat) of 2.97 min(-1), while CHR 745 had a K(m) of 7.80 microM and a K(cat) of 5.7 min(-1). The turnover numbers (K(cat)/K(m)) of CHR 859 and CHR 745 were 1.9 x 10(6) M min(-1) and 7.4 x 10(5) M min(-1), respectively, indicating CHR 859 is 2.6 times more efficient than CHR 745 in destroying CTGF mRNA. Stable transfection of CHR 859 into human fibroblasts reduced CTGF mRNA levels 55% and protein levels 72% compared to the inactive ribozyme control. Furthermore, TGF-beta-induced cell proliferation was reduced 90% in fibroblasts stably transfected with CHR 859 compared to control cell groups.


CONCLUSIONS
The CHR 859 hammerhead ribozyme cleaved human CTGF mRNA with high kinetic efficiency in vitro, effectively reduced levels of CTGF mRNA and protein in cultured human fibroblasts, and blocked TGF-beta-induced cell proliferation without nonspecific toxicity. These data support the concept that CTGF mediates TGF-beta-induced cell proliferation, and imply that regulating CTGF expression with ribozymes may be effective in reducing ocular scarring.
Blalock
Timothy D
TD
Department of Ob/Gyn, College of Medicine, Institute for Wound Research, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0294, USA. tablalock@obgyn.ufl.edu
Yuan
Rong
R
Lewin
Alfred S
AS
Schultz
Gregory S
GS
D001483:Base Sequence; D002384:Catalysis; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C427204:hammerhead ribozyme; D055513:Connective Tissue Growth Factor