Interaction between Periostin and BMP-1 Promotes Proteolytic Activation of Lysyl Oxidase

Takumi Maruhashi,Isao Kii,Mitsuru Saito,Akira Kudo

doi:10.1074/jbc.m109.088864

Abstract

Intra- and intermolecular covalent cross-linking between collagen fibrils, catalyzed by lysyl oxidase (LOX), determines the mechanical properties of connective tissues; however, mechanisms that regulate the collagen cross-linking according to tissue specificity are not well understood. Here we show that periostin, a secretory protein in the dense connective tissues, promotes the activation of LOX. Previous studies showed that periostin null mice exhibit reduced collagen cross-linking in their femurs, periosteum, infarcted myocardium, and tendons. Presently, we showed that active LOX protein, formed by cleavage of its propeptide by bone morphogenetic protein-1 (BMP-1), was decreased in calvarial osteoblast cells derived from periostin null mice. Overexpression of periostin promoted the proteolytic cleavage of the propeptide, which increased the amount of active LOX protein. The results of co-immunoprecipitation and solid phase binding assays revealed that periostin interacted with BMP-1. Furthermore, this interaction probably resulted in enhanced deposition of BMP-1 on the extracellular matrix, suggesting that this enhanced deposition would lead to cleavage of the propeptide of LOX. Thus, we demonstrated that periostin supported BMP-1-mediated proteolytic activation of LOX on the extracellular matrix, which promoted collagen cross-linking.

Highlights

Enzyme lysyl oxidase (LOX).2 The strength of connective tissues is determined by the amount of total collagen cross-linking, as well as by the total collagen content [2,3,4]
RT-PCR analysis showed no significant difference in the expression of LOX family genes between the WT and periostinϪ/Ϫ periosteum (Fig. 1A), indicating that the reduced crosslinking of collagens in the periostinϪ/Ϫ mice might possibly be caused by decreased activity of LOX proteins
We demonstrate that periostin bound to BMP-1 and promoted the proteolytic activation of pro-LOX

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Rabbit anti-mouse periostin antibodies (antiRD1) were described previously [14]. Specific primer sets for each LOX family gene, periostin, BMP-1, and glyceraldehyde-3-phosphate dehydrogenase were designed, and the primer sequences used were the following: LOX, 5Ј-GTTCCAAGCTGGTTTCTCGC-3Ј (sense) and antisense 5Ј-CTGGATGTAGTAGGGGTCGG-3Ј (antisense) (annealing temperature, 63 °C; number of cycles: 29 for periosteum, for COB cells, and for 10T1/2 transfectants); lysyl oxidase-like 1 protein (LOXL1), 5Ј-GCAT-. After concentration of total proteins using a Microcon Ultracel YM-10 (Millipore, Billerica, MA), the cell culture supernatants were added with SDS sample buffer containing 50 mM dithiothreitol. The 293T transfectants stably expressing BMP-1-FLAG were grown to confluence, washed with PBS, and scraped into lysis buffer (50 mM HEPES-NaOH, pH 7.4, containing 150 mM NaCl and 0.5% Nonidet P-40) on ice. The clarified lysate was loaded onto an anti-FLAG antibody-conjugated agarose chromatography column (Sigma-Aldrich). Statistical significance was assessed by use of the unpaired t test (p values Ͻ 0.05 were considered significant)

RESULTS

Overexpression of Periostin Increases the Amount of Active

DISCUSSION