Elsevier

Bone

Volume 40, Issue 2, February 2007, Pages 305-315
Bone

Osteoclast inhibitory lectin (OCIL) inhibits osteoblast differentiation and function in vitro

https://doi.org/10.1016/j.bone.2006.09.001Get rights and content

Abstract

Osteoclast inhibitory lectin (OCIL) is a type II C-type lectin and binds NK cell-associated receptor Nkrp1d and sulfated glycosaminoglycans. OCIL is expressed by several cell types found in bone and inhibits osteoclast differentiation. To determine whether OCIL may have wider effects on bone metabolism, we examined the effects of recombinant soluble OCIL on cultured osteoblasts and pre-osteoblastic KUSA O cells. Although OCIL did not affect osteoblast proliferation or apoptosis, or the formation of alkaline phosphatase positive colonies in cultured bone marrow, OCIL profoundly inhibited mineralization by primary osteoblasts and KUSA O cells in vitro. Analysis of ascorbate-treated KUSA O cells showed that addition of OCIL reduced bone sialoprotein (BSP), osterix and osteocalcin mRNA expression, as well as alkaline phosphatase activity while, in contrast, expression of markers associated with the earlier stages of osteoblast maturation or the transcription factors Runx2, ATF4 and c-fos were not affected by OCIL treatment. Indeed, osteocalcin expression was strongly inhibited within 3 days in a dose-dependent manner, although after subsequent removal of OCIL, osteocalcin mRNA levels recovered within 4 days. OCIL treatment also reduced osteocalcin expression in BMP-2 stimulated C2C12 cells. In support of a role for OCIL in mineralization, OCIL anti-sense oligonucleotide treatment of KUSA O cells increased mineralization and osteocalcin expression. In addition, insulin-, dexamethasone- and IBMX-stimulated KUSA O cells undergo adipocyte differentiation and OCIL treatment greatly suppressed this process. Consistent with this, OCIL also reduced adiponectin and resistin mRNA expression in these cells. Our data indicate that OCIL reduces osteoblastic function in vitro and this may be due to an inhibitory effect on osteoblast maturation. In addition, the reduction of adipocyte formation in KUSA O cells by OCIL indicates that OCIL may have wider effects on the mesenchymal lineage that may be important for both bone metabolism and other connective tissue functions.

Introduction

OCIL is a 207 amino acid type II membrane bound C-type lectin that was originally identified in murine osteoblasts, where its expression is regulated by osteolytic hormones [1]. OCIL mRNA and protein expression has been identified in hematopoietic, epithelial and mesenchymal cell populations [2]. Soluble recombinant forms of the extracellular domain of murine OCIL, including fusion proteins produced in E. coli (GST-OCIL76–206 and MBP-OCIL76–206) and mammalian cells (FLAG-OCIL76–206), inhibit mouse and human osteoclast formation in vitro[1], [2]. A human homolog as well as two closely related murine proteins, OCILrP1 and OCILrP2, has been identified and recombinant soluble extracellular domains of each of these proteins inhibit osteoclast formation [2], [3]. OCILrP2 (but not OCILrP1) is expressed by osteoblasts although, unlike OCIL, its expression is not regulated by osteolytic hormones [2], [3]. The patterns of cellular expression of these proteins and their effects on osteoclast progenitors suggest a role in bone metabolism, with perhaps some redundancy of function. Such a role may also be suggested by the high affinity of OCIL for large sulfated glycosaminoglycans (GAG) (including heparan sulfate and chondroitin sulfate) that are abundant in the bone microenvironment, although carbohydrate binding does not affect anti-osteoclastogenic actions of OCIL [4]. OCIL action may be mediated by its recently identified receptor, Nkrp1d, which is also a C-type lectin and is expressed by natural killer (NK) cells [5], [6]. Indeed, like the Nkrp1d gene, the ocil gene is located in the NK cell gene complex on mouse chromosome 6 [5], [7], [8] as are genes for other C-type lectins such as CD69 and other Nkrp1 family members. Nkrp1d-OCIL binding plays a role in NK cell activation and killing [5], [6]; however, the role of Nkrp1 family members in mediating other actions of OCIL is unknown.

Since OCIL expression is detectable in numerous cell types found in the bone microenvironment (including hematopoietic lineage cells and osteoclasts) and, since osteoblast and osteoclast action are closely coupled, it is possible that OCIL also exerts some effects on the osteoblast lineage. However, while many C-type lectins have been extensively investigated in lymphocytes and the immune system, none have been studied for their effects on connective tissues or mesenchymal-derived cells. We therefore set out to determine whether OCIL acts on cells of the osteoblastic lineage by examining the effects of OCIL treatment in primary murine osteoblasts as well as a bipotential osteoblast/adipocyte forming cell line, KUSA O [9].

Section snippets

Reagents and cell culture

Cells were cultured in alpha minimal essential medium (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum (CSL Biosciences, Parkville, Australia) (MEM/FBS), apart from C2C12 differentiation assays which were performed in minimal essential medium containing 2% horse serum (CSL Biosciences) (MEM/HS). Recombinant soluble proteins GST-OCIL76–206 (OCIL), GST, MBP-OCIL76–206 and MBP were prepared as previously described [1], [4]. Biochemical reagents were analytical grade and obtained

The influence of OCIL on osteoblast and KUSA O cell mineralization in vitro

Primary cultured calvarial osteoblasts (Fig. 1A) and KUSA O cells (Fig. 1B) both formed numerous mineralization nodules when stimulated for 21 days in the presence of ascorbate and β-glycerophosphate, as previously described [10]. Recombinant OCIL (500 ng/ml) strongly suppressed mineralization by both osteoblasts and KUSA O cells (Figs. 1A, B) compared to positive control cultures. Since the recombinant OCIL protein employed was a GST fusion protein, mineralization cultures treated with GST

Discussion

We have found that the soluble recombinant form of the extracellular domain of the membrane bound C-type lectin OCIL is able to block osteoblastic and pre-adipocytic cell differentiation in vitro, providing the first demonstration of an activity of an NK C-type lectin on mesenchymal-derived stromal cells. We have recently investigated the effects of ablating the ocil gene in mice in vivo (manuscript in preparation, and [28]). The ocil−/− mice have approximately 20% less trabecular bone volume

Acknowledgments

This work was supported by Program Grant (345401) from the National Health and Medical Research Council of Australia. We thank Patricia Smith for help in the production of purified recombinant proteins.

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