Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells

BACKGROUND: Recent reports suggest that thymosin beta-4 (Tβ4) is a key regulator for wound healing and anti-inflammation. However, the role of Tβ4 in osteoclast differentiation remains unclear. PURPOSE: The purpose of this study was to evaluate Tβ4 expression in H(2)O(2)-stimulated human periodontal ligament cells (PDLCs), the effects of Tβ4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. METHODS: Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H(2)O(2)-treated PDLCs. RESULTS: Tβ4 was down-regulated in H(2)O(2)-exposed PDLCs in dose- and time-dependent manners. Tβ4 activation with a Tβ4 peptide attenuated the H(2)O(2)-induced production of NO and PGE(2) and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tβ4 peptide inhibited the effects of H(2)O(2) on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tβ4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H(2)O(2) up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tβ4 on H(2)O(2)-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. CONCLUSION: In conclusion, this study demonstrated, for the first time, that Tβ4 was down-regulated in ROS-stimulated PDLCs as well as Tβ4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro. Thus, Tβ4 activation might be a therapeutic target for inflammatory osteolytic disease, such as periodontitis.