Establishment of a recombinant expression system for connective tissue growth factor (CTGF) that models CTGF processing in utero.

Connective tissue growth factor (CTGF) stimulates cell proliferation, migration, adhesion and extracellular matrix production, and functions in processes such as development, differentiation, angiogenesis, implantation, wound healing and fibrosis. CTGF is a 38 kDa protein that comprises four discrete structural modules (modules 1-4) but is susceptible to limited proteolysis in utero yielding bioactive isoforms that comprise either modules 3 and 4 (16-20 kDa) or module 4 (10 kDa). Here we report the development of a stable cell line, termed DB1, that was generated by transfecting cDNA encoding full-length human CTGF into Chinese hamster ovary cells that were mutant for heparin sulphate and chondroitin sulphate. DB1 cells produced 38 kDa CTGF and low molecular mass CTGFs that had N-termini between modules 2 and 3 at Ala(181) (20 kDa), Leu(184) (18 kDa) or Ala(197) (16 kDa) or between modules 3 and 4 at Gly(253) (10 kDa). CTGF was exported from DB1 cells as early as 5 min after synthesis and all isoforms were readily purified from conditioned medium by sequential steps of heparin affinity, cation exchange, and reverse-phase chromatography. The 38 kDa CTGF was faithfully glycosylated and underwent limited proteolysis in the presence of thrombin, kallikrein or uterine fluids, the last of which was antagonized by anti-thrombin III. All CTGF isoforms promoted cell adhesion, mitosis and epithelial transdifferentiation in vitro as well as subcutaneous fibrosis in vivo. The establishment of this recombinant expression system allows for mass-scale production of all previously reported uterine CTGF isoforms, demonstrates that module 4 contains functional domains involved in a broad range of biological activities, and will facilitate studies of CTGF processing in vitro.

The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion.

Connective tissue growth factor (CTGF) is a 349-residue mosaic protein that contains four structural modules implicated in protein-protein interactions. To address the functionality of residues 247-349 (containing module 4 alone), this region of CTGF was produced as a maltose binding protein (MBP) fusion protein in E. coli. After removal of MBP, recombinant CTGF commenced at Glu(247), was of M(r) 10 000, was immunoreactive with anti-CTGF[247-260], bound strongly to heparin, and promoted dose-dependent adhesion of fibroblasts, myofibroblasts, endothelial cells, and epithelial cells. An 8 kDa presumptive C-terminally truncated form of CTGF commencing at Glu(247) also promoted cell adhesion. CTGF-mediated cell adhesion was abolished by heparin or EDTA. These data demonstrate the presence of heparin-binding and cell-adhesion motifs within the C-terminal 103 residues of CTGF and show that CTGF-mediated cell adhesion is heparin-and divalent cation-dependent. Thus, CTGF isoforms comprising essentially module 4 are intrinsically functional in the absence of the other constituent modules of CTGF.