The role of haptoglobin in osteoclastogenesis

Abstract

Haptoglobin (Hp), a member of the acute phase proteins, is known to be a major hemoglobin (Hb)-binding protein that plays a protective role against Hb-induced cytotoxicity in various organs. Hp is primarily expressed in hepatocytes, and recent studies have shown its expression in other cells, such as keratinocytes, leucocytes, fibrocytes and adipocytes. However, the involvement of Hp in bone-related cells has not been fully understood. In this study, I investigated the effects of Hp in osteoclastogenesis and skeletal health. Inflammatory cytokines, interleukin-1α (IL-1α), tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS) induced Hp secretion in the bone-forming cells, osteoblasts. Histo-morphometric analyses indicated that the deletion of Hp gene showed significant bone loss with increasing osteoclast formation. Administration of Hp in Hp knockout (Hp-/-) mice stimulated a higher bone volume increment than that in PBS-injected mice. Consistent with the in vivo results, IL-1α-induced osteoclast formation by co-culture of Hp null calvarial osteoblast and wild-type (WT) bone marrow-derived macrophages (BMMs) showed increase higher osteoclastogenesis than that observed in WT osteoblast in vitro. Stimulation of Hp inhibited osteoclastogenesis by suppression of major transcription factors such as c-Fos and NFATc1 expression. Overexpression of c-Fos in osteoclast precursor cells rescued Hp-mediated suppression of osteoclastogenesis with promotion of its down-stream master transcription factor, NFATc1. I found that Hp-induced suppression of c-Fos expression was mediated by an increase in interferon beta (IFNβ) levels, a well-known c-Fos inhibitor. Hp-induced inhibition of osteoclastogenesis substantially recovered following treatment with IFNβ-specific neutralizing antibody and IFN-type I receptor knockout cells. In addition, I determined that Hp-induced IFNβ expression was activated via toll-like receptor 4 (TLR4). Flow cytometer analysis showed that stimulation of Hp or the well-known TLR4 ligand, LPS induced TLR4 internalization. Furthermore, a binding assay for TLR4 showed direct interaction of Hp to TLR4 when I compared to that of bovine serum albumin (BSA) interaction. Taken together, these results demonstrate that inflammation-induced secreted-Hp from osteoblast plays a protective role against excessive osteoclastogenesis via the TLR4-IFNβ signaling pathway.