Osteoclast inhibitory lectin (OCIL) inhibits osteoblast differentiation and function in vitro
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.
References (34)
- et al.
A novel osteoblast-derived c-type lectin that inhibits osteoclast formation
J. Biol. Chem.
(2001) - et al.
Osteoclast inhibitory lectin—A family of new osteoclast inhibitors
J. Biol. Chem.
(2002) - et al.
Characterization of sugar binding by osteoclast inhibitory lectin
J. Biol. Chem.
(2004) - et al.
Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP)
J. Biol. Chem.
(1995) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
Modulation of the signal recognition particle 54-kDa subunit (SRP54) in rat preosteoblasts by the extracellular matrix
J. Biol. Chem.
(1995) - et al.
Post-transcriptional regulation of the human liver/bone/kidney alkaline phosphatase gene
J. Biol. Chem.
(1991) - et al.
Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation
Cell
(1997) - et al.
The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation
Cell
(2002) - et al.
The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function
J. Biol. Chem.
(2000)
Glucocorticoids inhibit developmental stage-specific osteoblast cell cycle. Dissociation of cyclin A-cyclin-dependent kinase 2 from E2F4–p130 complexes
J. Biol. Chem.
Isolation of a human homolog of osteoclast inhibitory lectin that inhibits the formation and function of osteoclasts
J. Bone Miner. Res.
Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors
Nat. Immunol.
Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1 natural killer cell receptors
Proc. Natl. Acad. Sci. U. S. A.
Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69
Immunogenetics
Immune functions encoded by the natural killer gene complex
Nat. Rev., Immunol.
Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo
J. Cell. Physiol.
Cited by (32)
Molecular signaling in bone cells: Regulation of cell differentiation and survival
2019, Advances in Protein Chemistry and Structural BiologyCitation Excerpt :Estrogens act via a complex signaling mechanism to inhibit the activation of T cells, decreasing their secretion of RANKL and TNFα. Estrogen also inhibits osteoclast differentiation and decreases life span, by regulating the expression of Fas ligand (FasL) in osteoblasts (Krum et al., 2008), which binds the death receptor Fas in osteoclasts (Nakamura, Imai, et al., 2007; Nakamura, Ly, et al., 2007), accelerating osteoclast cell death. Finally, the anti-resorptive amino-bisphosphonates also decrease bone remodeling by decreasing osteoclast activity and by inducing osteoclast apoptosis (Dunford, Rogers, Ebetino, Phipps, & Coxon, 2006; Tella & Gallagher, 2014; Tsubaki et al., 2014).
Multi-functional lectin-like transcript-1: A new player in human immune regulation
2016, Immunology LettersEGFL6 promotes endothelial cell migration and angiogenesis through the activation of extracellular signal-regulated kinase
2011, Journal of Biological ChemistryCitation Excerpt :We next investigated the effect of EGFL6 on bone mineralization in KusaO cells using alizarin red staining assay. KusaO cell is a mouse preosteoblast cell line established for studying osteoblast differentiation (34, 35). As shown in Fig. 7B, mineralization area assessments by alizarin red staining were performed after 14 days of culture with the conditioned medium containing EGFL6 or vehicle control under differentiation condition.
Inhibitory C-type lectin receptors in myeloid cells
2011, Immunology Letters