Purification and Characterization of Novel Heparin-binding Growth Factors in Uterine Secretory Fluids

Abstract

Uterine growth factors are potential effector molecules in embryo growth signaling pathways. Pig uterine luminal flushings contained a heparin-binding growth factor (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8. This factor, which was heat-and acid-labile and of M r 10,000 as assessed by gel filtration, stimulated DNA synthesis in fibroblasts and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent M r of 10,000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EEN-IKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKX-IRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247-262 and 248 -259 of the 349-residue predicted primary translation product of porcine connective tissue growth factor (pCTGF). 10-kDa CTGF-mediated fibroblast DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and 20 kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel growth factor in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.

Connective Tissue Growth Factor (CTGF) Is Regulated by Wnt and Bone Morphogenetic Proteins Signaling in Osteoblast Differentiation of Mesenchymal Stem Cells

“…Module IV resembles the Cterminal cystine-knot domain of several extracellular proteins including von Willebrand factor and mucins. Sequence similarities to heparin-binding motifs are also found within this domain (69). In CTGF, the C-terminal cystine knot domain is sufficient to mediate cell adhesion in a divalent cation-and heparin-dependent manner (70).…”

Kallikrein-related Peptidase 12 Hydrolyzes Matricellular Proteins of the CCN Family and Modifies Interactions of CCN1 and CCN5 with Growth Factors

“…Various forms of CCN2 were detected in pig uterine flushings resulting from in utero proteolytic digestion of the full-length protein (41)(42)(43). In rats, an N-terminally truncated CCN3 form predominates in brain tissue and in cerebrospinal fluid (44).…”

Heparin affin regulatory peptide binds to vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis

“…This protein, initially identified in the conditioned media of HUVEC (Bradham et al, 1991), is characterized by a modular structure with four distinct domains including insulin-like growth factor binding protein, von Willebrand factor type C, TSR-1 repeat, and COOH-terminal domains (Bork, 1993). Several studies indicated that CTGF is mitogenic and a chemotactic factor for cultured fibroblasts that has been implicated in wound healing (Igarashi et al, 1993), fibrotic disorders (Igarashi et al, 1996), and uterine function (Brigstock et al, 1997). Further studies described that CTGF displayed mitogenic activities for endothelial cells (Shimo et al, 1998) and promoted angiogenesis (Shimo et al, 1999).…”