Apelin and its receptor are expressed in human osteoblasts
Introduction
It has been demonstrated that body fat and bone mass are directly related, and many studies have shown a positive association of bone mineral density (BMD) with fat mass [1], [2], [3], [4], [5], [6]. Although recent studies showed that leptin and adiponectin secreted by adipocytes contribute to this relationship, other factors may be also involved.
Apelin is produced in a variety of tissues and is shown also to be a new adipokine secreted from the adipocytes and is present in plasma [7], [8], [9], [10], [11], [12], [13], [14]. Apelin is up-regulated by obesity [13]. Apelin was found in 1998 to be an endogenous ligand of the human orphan G protein-coupled receptor APJ, which previously had been identified by the Human Genome Project in 1993 as a member of the superfamily of 7-transmembrane G-protein coupled receptors [11], [15]. Apelin and APJ are widely expressed throughout the body [9], [14], [16], [17]. This peptide is produced through processing from the C-terminal portion in the pre-proprotein consisting of 77 amino acid residues and exists in multiple molecular forms [11]. In all species cloned to date, a sequence of 23 amino acids in the C-terminal region is conserved, implying that the C-terminal region is critical for biological activity [10]. Before the isolation of apelin, the APJ receptor was referred to as an orphan receptor because its endogenous ligand was unidentified. APJ mediated the biological effects of apelin [18]. It has been reported that apelin peptides bind APJ receptor and promote receptor internalization, and inhibit cAMP formation and activate MAP kinase and PI3 kinase pathways [19], [20], [21], [22], [23].
To date, there is no information on the expression of apelin and APJ in osteoblasts, nor is there any information on the effects of apelin in osteoblasts. The purpose of the present study was to characterize apelin and apelin receptor APJ expression in normal human osteoblasts, and to assess the effects of apelin on osteoblast proliferation and differentiation.
Section snippets
Reagents
Synthetic apelin-13 peptide was purchased from American Peptide Company Inc. (Sunnyvale, CA, USA). The amino acid sequence of apelin-13 is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe. Recombinant human IGF-II protein was purchased from PEPROTECH (London, UK). Anti-extracellular signal-regulated kinase1/2 (ERK1/2), p-ERK, p38, p-p38, c-jun N-terminal Kinase (JNK), p-JNK, Akt, p-Akt antibody, anti-mouse, and rabbit IgG peroxidase conjugate antibodies were purchased from Santa Cruz
Characterization of human osteoblast-like cells in vitro
Cells obtained from collagenase-digested human trabecular bone were characterized as osteoblast-like cells by several criteria, including high intrinsic alkaline phosphatase (ALP) activity, secretion of osteocalcin and type I procollagen, and cAMP response to parathyroid hormone (PTH). ALP levels in normal human osteoblasts were 79.8 ± 5.6 nmol/min mg protein. Radioimmunoassay (RIA) of culture supernatants from unstimulated human bone cell cultures revealed detectable osteocalcin levels (4.24 ± 0.43
Discussion
Apelin interacts with its specific receptor APJ, has multiple biological activities, and had been characterized in various tissues [7], [8], [9], [10], [11], [12], [13], [19], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50]. However, the expression of apelin and APJ in human osteoblasts and the effects of apelin/APJ on human osteoblasts remain unknown. The present study demonstrates the transcription and translation of apelin and APJ from primary human osteoblasts in vitro. We
Acknowledgements
This work was supported by Grant-30200322, 30400218 from China National Natural Scientific Foundation, A Foundation for the Author of National Excellent Doctoral Dissertation of PR China (200259), and Hunan Provincial Outstanding Younger Foundation of PR China (03JJY1005).
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