Connective Tissue Growth Factor Enhances Osteoblastogenesis in Vitro

Anna Smerdel-Ramoya,Stefano Zanotti,Valerie Deregowski,Ernesto Canalis

doi:10.1074/jbc.m710140200

Abstract

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage has not been established. We investigated the effects of CTGF overexpression by transducing murine ST-2 stromal cells with a retroviral vector, where CTGF is under the control of the cytomegalovirus promoter. Overexpression of CTGF in ST-2 cells increased alkaline phosphatase activity, osteocalcin and alkaline phosphatase mRNA levels, and mineralized nodule formation. CTGF overexpression decreased the effect of bone morphogenetic protein-2 on Smad 1/5/8 phosphorylation and of Wnt 3 on cytosolic beta-catenin, indicating that the stimulatory effect on osteoblastogenesis was unrelated to BMP and Wnt signaling. CTGF overexpression suppressed Notch signaling and induced the transcription of hairy and E (spl)-1 (HES)-1, by Notch-independent mechanisms. CTGF induced nuclear factor of activated T cells (NFAT) transactivation by a calcineurin-dependent mechanism. Down-regulation of CTGF enhanced Notch signaling and decreased HES-1 transcription and NFAT transactivation. Similar effects were observed following forced CTGF overexpression, the addition of CTGF protein, or the transduction of ST-2 cells with a retroviral vector expressing HES-1. In conclusion, CTGF enhances osteoblastogenesis, possibly by inhibiting Notch signaling and inducing HES-1 transcription and NFAT transactivation.

Highlights

Experiments conducted in ctgf null calvarial osteoblasts or in C3H10T 1⁄2 cells following the down-regulation of connective tissue growth factor (CTGF) using RNA interference (RNAi) have demonstrated that CTGF is necessary for osteoblastogenesis [10, 13]
To examine the impact of CTGF on the ST-2 cell phenotype, ST-2 cells were transduced with the retroviral expression construct pLPCX-CTGF and compared with cells transduced with pLPCX vector
To study the mechanisms involved in the induction of nuclear factor of activated T cells (NFAT) by hairy and E (spl)-1 (HES)-1, we examined for potential interactions between HES-1 and the related HES related protein (HERP)-2, which is known to form heterodimers with HES-1 and to inhibit osteoblastogenesis [29, 49]

Summary

Introduction

2 The abbreviations used are: BMP, bone morphogenetic protein; APA, alkaline phosphatase activity; CMV, cytomegalovirus; CR, cysteine-rich; CSL, CBF1/Suppressor of Hairless/Lag 1; CTGF, connective tissue growth factor; Cyr 61, cysteine-rich 61; EMSA, electrophoretic mobility shift assay; ERK, extracellular regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HES-1, hairy and E (spl)-1; HERP, HES-related repressor osteoblastogenesis [2,3,4]. Other signals, such as Notch, impair the differentiation of cells of the osteoblastic lineage [5]. The structural similarities with BMP antagonists of the Tsg and chordin families and these observations indicate a potential functional relationship between CCN peptides and extracellular BMP antagonists [6]

Objectives

Results

Conclusion