TGF-β Inducible Early Gene 1 Regulates Osteoclast Differentiation and Survival by Mediating the NFATc1, AKT, and MEK/ERK Signaling Pathways

Muzaffer Cicek,John R Hawse,Malayannan Subramaniam,Catherine Sturchio,Thomas C Spelsberg,Merry Jo Oursler,Larry Pederson,Anne Vrabel,Dhyan Chandra

doi:10.1371/journal.pone.0017522

Abstract

TGF-β Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG1−/− mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1−/− osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1−/− precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1−/− osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1−/− osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1−/− cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1−/− precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.

Highlights

During osteoclast formation, RANKL and M-CSF activate NFkB, c-Jun N-terminal kinase, ERK, and AKT [1,2,3,4,5,6]
Loss of TGF-b Inducible Early Gene-1 (TIEG1) Delays Osteoclast Differentiation in Vitro We compared the ability of WT and TIEG12/2 marrowderived osteoclast precursors to differentiate in vitro into osteoclasts when treated with M-CSF and RANKL (Figure 1)
To ensure that there were no contaminating mesenchymal cells in our experiments which could contribute to the observed differences in osteoclast differentiation, we cultured the non-adherent cells from WT and TIEG12/2 marrow in the absence of MCSF

Summary

Introduction

During osteoclast formation, RANKL and M-CSF activate NFkB, c-Jun N-terminal kinase, ERK, and AKT [1,2,3,4,5,6]. These signaling pathways modulate osteoclast survival in response to RANKL and M-CSF. NFATc1 is considered a master regulator of RANKL-induced osteoclastogenesis since reduced expression of NFATc1 causes defects in osteoclastogenesis in response to RANKL. NFATc1 is regulated by the serine/threonine phosphatase calcineurin, which is activated by intracellular Ca2+. The mechanisms by which these diverse proteins function remain mostly unresolved [13]

Methods

Results

Conclusion