The NH2-terminal Propeptide of Type I Procollagen Acts Intracellularly to Modulate Cell Function

Abstract

The function of the NH 2 -terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-␤1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (␣1 trimer) during vertebrate development.Fibrillar types I-III procollagens are proteolytically cleaved by procollagen N-and C-proteases that separate nontriple helical NH 2 -and COOH-terminal propeptides, respectively, from their major central triple helical domains (1). This proteolytic processing is necessary for the alignment and self-assembly of collagen molecules and for normal collagen fibril formation. Specific sequences in the COOH-terminal propeptides are required for chain recognition and triple helix formation that generate procollagens with the correct chain composition (2-4). In addition, the COOH-terminal propeptide has been shown to be chemotactic for endothelial cells and to stimulate migration and production of metalloproteinases by mammary carcinoma cell lines after its release by procollagen C protease (5, 6). Fragments of the propeptide have also been reported to stimulate extracellular matrix production (7).In contrast, the functions of the NH 2 -terminal propeptide of type I procollagen (N-propeptide) 2 are not well understood (for a review see Ref. 8). In 1979, Wiestner et al. (9) purified the N-propeptide from dermatosparactic calf skin, which is deficient in procollagen N-protease, and demonstrated that it functioned to inhibit the synthesis of both types I and III collagen by bovine skin fibroblasts in culture. Subsequently, evidence was provided for the ability of the N-propeptide to inhibit selectively the translation of procollagen mRNA in cell-free systems (10, 11). However, this function, which presumes a cytosolic site of action, was never reconciled with the fact that the N-propeptide is released only in intracellular secretory vesicles and in the extracellular environment (12). Additional functions proposed for the N-propeptide included inhibiti...

Diminished Type III Collagen Promotes Myofibroblast Differentiation and Increases Scar Deposition in Cutaneous Wound Healing

“…The N-propeptides of both type I and II collagen, which show close sequence homology with that of Col3, have previously been shown to modulate TGF ␤ family member signaling [Zhu et al, 1999;Oganesian et al, 2006], suggesting a potential mechanism for the regulation of TGF ␤ activity by Col3. Regulation of the TGF ␤ isoform's bioavailability is of particular interest due to its key role in the regulation of the myofibroblast phenotype and scar tissue formation [Desmouliere et al, 1993;Vaughan et al, 2000;Gabbiani, 2003].…”

Type IIB Procollagen NH2-propeptide Induces Death of Tumor Cells via Interaction with Integrins αVβ3 and αVβ5

“…The other fibrillar collagens, types I, III, and V, are also synthesized with NH 2 -and COOH-propeptides that are removed prior to deposition of fibrils in the ECM. Function has been attributed to the liberated type I NH 2 -propeptide, which has been suggested to be involved in feedback inhibition of collagen synthesis (44) and, based on the similarity to type IIA procollagen NH 2 -propeptide, has recently been shown to bind to BMP2 and transforming growth factor-␤1 and to act intracellularly (45). This mechanism has nothing to do with integrin binding but functions via the von Willebrand factor C domain of type I and type IIA collagens.…”

Osteogenesis imperfecta

“…The resulting pN collagen (that is, the mature collagen with the N propeptide attached) assembles into small diameter fibrils with regular borders 39,40 . Retention of the N propeptide in the collagen molecule might also compromise the intracellular functions of the pro peptide, such as protein phosphorylation, collagen synthesis and cell adhesion 41,42 . Furthermore, the N propeptide in the extracellular matrix binds to specific cytokines (for example, transforming growth factor β1 (TGFβ1) and BMP2), which are crucial for bone development 42 .…”

“…Retention of the N propeptide in the collagen molecule might also compromise the intracellular functions of the pro peptide, such as protein phosphorylation, collagen synthesis and cell adhesion 41,42 . Furthermore, the N propeptide in the extracellular matrix binds to specific cytokines (for example, transforming growth factor β1 (TGFβ1) and BMP2), which are crucial for bone development 42 . Mutations that remove the N terminal cleavage site residues due to exon skipping 43 cause Ehlers-Danlos syndrome, and not osteogenesis imperfecta…”