Synergism between Wnt3a and Heparin Enhances Osteogenesis via a Phosphoinositide 3-Kinase/Akt/RUNX2 Pathway

Ling Ling,Christian Dombrowski,Kin Mun Foong,Larisa M Haupt,Gary S Stein,Victor Nurcombe,Andre J Van Wijnen,Simon M Cool

doi:10.1074/jbc.m110.122069

Abstract

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Highlights

Roid hormone, and Wnts) is limited, and new molecular approaches to improve the effectiveness and selectivity of osteo-inductive extracellular ligands are desirable
Stimulation of the canonical Wnt pathway in response to Wnt3a was reflected by a dose-dependent increase in the transcriptional activity of lymphoid enhancer binding factor 1 (LEF1)/T cell-specific factor (TCF) that reaches maximal potency at 50 ng/ml as measured by transient transfection assays with the TOP-FLASH reporter in MC3T3-E1 cells (Fig. 1A)
The ineffectiveness of Wnt3a in stimulating ALP in MC3T3-E1 osteoblasts was notable considering that the dose we applied (50 ng/ml) was optimal for the transcriptional activation of ␤-catenin-LEF1/TCF complexes and nuclear translocation (Fig. 1, A and B)

Summary

Introduction

Roid hormone, and Wnts) is limited, and new molecular approaches to improve the effectiveness and selectivity of osteo-inductive extracellular ligands are desirable. Stimulation of the canonical Wnt pathway in response to Wnt3a was reflected by a dose-dependent increase in the transcriptional activity of LEF1/TCF that reaches maximal potency at 50 ng/ml (within the dose range tested) as measured by transient transfection assays with the TOP-FLASH reporter in MC3T3-E1 cells (Fig. 1A).

Results

Conclusion