Biochemical and Biophysical Research Communications
Knockdown of Lrp1 in RAW264 cells inhibits osteoclast differentiation and osteoclast-osteoblast interactions in vitro
Introduction
Bone homeostasis is maintained through the coordination of bone resorption by osteoclasts and bone formation by osteoblasts [1,2]. Since imbalance between bone resorption and formation results in osteoporosis or osteopetrosis [3], mechanisms of osteoclast differentiation and osteoclast-osteoblast communication are consequential in the pathogenesis of bone metabolic diseases. Osteoclasts are differentiated from monocyte/macrophage-lineages stimulated by Receptor Activator of nuclear factor-κB (NFκB) Ligand (RANKL) [4], and stimulate osteoblastic bone formation by humoral factors [2,5]. These factors are secreted by osteoclasts, released from the bone matrix during bone resorption, or expressed on the cell surface membrane of osteoclasts, serving as coupling factors of bone resorption and formation [2,5,6]. On the other hand, it has also been reported that osteoclast-secreted Semaphorin 4D and Sclerostin serve as inhibitors of bone formation [7,8]. These evidences suggest that osteoclasts play bifunctional roles in mediating osteoblastic bone formation.
Low density lipoprotein receptor-related protein 1 (LRP1; also known as CD91), a member of the low-density lipoprotein receptor family, functions as a receptor for several ligands such as α-2 macroglobulin, Apolipoprotein E and tissue plasminogen activator [9,10]. In human study, LRP1 single nucleotide polymorphism (SNP) is associated with bone mineral density [11]. Because of the low bone mass phenotype in Ctsk-Cre mediated Lrp1 conditional knockout mice [12], LRP1 expressed in mature osteoclasts is thought to suppress osteoclast differentiation. Functions of LRP1 in early osteoclastic differentiation from macrophages are, however, not fully understood. In this study, we generated Lrp1 knockdown mouse macrophage cell line RAW264 cells by the lentiviral short hairpin RNA (shRNA) vector in order to gain insight into the function of Lrp1 in osteoclast precursors. Knockdown of Lrp1 in RAW264 cells significantly reduced osteoclastogenesis and osteoclast-osteoblast interactions in vitro, suggesting that LRP1 mediates osteoclastic differentiation and function.
Section snippets
Reagents
We purchased recombinant GST-RANKL (Cat# 47197900) from Oriental Yeast, and antibodies to LRP1 (Cat# ab92544) from Abcam; to Cathepsin K (Cat# sc-48353) from Santacruz Biotech; and to β-actin (Cat# 010–27841) from Wako. CycLex Cellular Bromo-2′-deoxy-uridine (BrdU) ELISA Kit Ver.2 (Cat# CY-1141V2) was purchased from MBL.
Immunohistochemistry
C57BL/6 J mice were purchased from Clea Japan and used according to the protocol approved by the Animal Experiment Committee of Ehime University, Japan. For the preparation of
Mature osteoclasts barely express LRP1 in vivo and in vitro
It has been recently reported that Ctsk-Cre mediated Lrp1 conditional knockout mice exhibit low bone mass due to the increased number of osteoclasts, suggesting that LRP1 suppresses osteoclast differentiation [12]. Whether or not mature osteoclasts per se express LRP1 in vivo is, however, still unclear. Therefore, we first asked whether Cathepsin K (encoded by the Ctsk gene)-positive mature osteoclasts express LRP1 in vivo. Double-labeling immunohistochemistry for Cathepsin K and LRP1 were
Conclusions
This study demonstrates that knockdown of Lrp1 suppresses differentiation into mature osteoclasts, cell proliferation, and inhibitory effects on osteoblast differentiation by osteoclastic humoral factors in macrophage cell line RAW264 cells. We propose that LRP1 is required for differentiation of macrophages into osteoclasts and mediates osteoclast-osteoblast interactions.
Declaration of competing interest
The authors declare no competing interests.
Acknowledgments
We thank Y. Takaoka and C. Shiraishi for technical assistance. This study was supported in part by JSPS KAKENHI grant numbers JP17K18438 (to Y.K.), and JP19K18514 (to Y.K.).
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