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

Abstract

Cartilage is resistant to tumor invasion. In the present study, we found that the NH 2 -propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH 2 -propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins ␣ v ␤ 5 and ␣ v ␤ 3 . Adhesion is abrogated by blocking with anti ␣ v ␤ 5 and ␣ v ␤ 3 antibodies. When ␣ v is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express ␣ v ␤ 3 and ␣ v ␤ 5 integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.Cartilage is a unique tissue in being composed of only one cell type, chondrocytes. In addition, cartilage is avascular and resistant to tumor invasion, although the mechanism by which this occurs is unknown. Cartilage ECM 2 is predominantly made up of fibrillar type IIB collagen and the large proteoglycan aggrecan. The fibrillar collagens, types I, II, III, V, and XI, are synthesized as procollagens containing NH 2 -and COOH-terminal extension peptides that are removed prior to deposition of the collagen monomers into fibrils in the extracellular matrix (1). During the chondrogenesis phase of endochondral bone development, large amounts of type IIB collagen are synthesized as the tissue is established. Thereafter, in the cartilaginous growth plate, the cells become hypertrophic, change their predominant collagen synthesis to type X collagen, and eventually die by apoptosis. The hypertrophic cartilage is vascularized and subsequently removed by specialized osteoclasts, and the tissue is replaced by bone synthesized by osteoblasts. By this process, the cartilage provides an anlagen for bone formation (2, 3).Type II procollagen is unique among the fibrillar collagens in containing vicinal RGD motifs in the NH 2 -terminal propeptide domain encoded by exon 6 of the COL2A1 gene (supplemental Fig. 1). RGD peptides serve as the primary integrin recognition sites in extracellular matrix proteins and, as such, play an important role in regulating cell/matrix interactions required for proper cell function. Integrins are cell adhesion molecules that consist of two non-covalently associated subunits ␣ and ␤ (4). Integrins are receptors for many ECM matrix proteins, such as for collagens (The binding of substrate to integrins on the cell surface stimulates intracellular signaling to affect gene expression (outside-in signaling), and the cell can alter the expression and affinity of integrins (inside-out signaling). This bidirectional signaling controls cellular activity, such as cell-cell and ce...

Antiangiogenic and anticancer molecules in cartilage

“…SPARC is a matricellular glycoprotein that is not only multifunctional, but is perhaps the only molecule here that has extensive reviews devoted to both its angiogenic (or tumourpromoting) and antiangiogenic (or tumourinhibiting) roles (Refs 172, 173, 174 194) that might have a role in keeping the cartilage avascular and could be exploited to kill tumours in vivo. The interaction of recombinant human PIIBNP (GST-PIIBNP) with hCh-1 cells (human chondrosarcoma cell line) was dependent on α V β 5 and α V β 3 integrins and took place in an RGDRGD-dependent manner in vitro.…”

Downregulation of Col1a1 induces differentiation in mouse spermatogonia

“…19 Moreover, the NH 2 -propeptide of the cartilage-characteristic collagen, such as type II B, PIIBNP, is released into the ECM prior to formation of the collagen fibrils, and when it is suppressed by small interfering RNA, adhesion is blocked. 20 Therefore, we chose the fragment (from 11 to 11036 bp) encoding the N-terminal of Col1a1 for antisense-mediated gene knockdown study.…”

Novel functions for Type II procollagen

“…These viscinal RGD's are conserved throughout mammalian species ( Figure 1A). Using recombinant PIIBNP and mutant PIIBNP (RGDRGD to RGARGA), we found that PIIBNP, but not mutPIIBNP was able to inhibit cell viability of a human chondrosarcoma cell line (hCh-1), a breast cancer cell line, MDA231, and HeLa cells (18). This inhibition was not dependent on the substrate supporting the cells indicating that the process was not simply anoikis in which the protein competes for integrin binding ( Figure 1B).…”

“…This inhibition was not dependent on the substrate supporting the cells indicating that the process was not simply anoikis in which the protein competes for integrin binding ( Figure 1B). We found that the hCh-1 cells were actually being killed by a process of programmed cell necrosis (18). Studies using a mouse model of breast cancer tumor development demonstrated that in vivo, injected PIIBNP could inhibit the growth of tumors ( Figure 1C, 18).…”

“…In the course of investigating the functional roles of the type II collagen NH 2 -propeptides, we found that recombinant PIIBNP was able to kill selected types of cells while PIIANP was never able to kill cells. Our initial studies focused on the ability of PIIBNP to kill tumor cells (18). Mutational analysis demonstrated that this activity was dependent on the presence of two RGD moieties located in the NH 2 -propeptide, that function to target adhesion to cells expressing the integrins avb3 and avb5.…”