Apelin and its receptor are expressed in human osteoblasts

https://doi.org/10.1016/j.regpep.2006.02.004Get rights and content

Abstract

Objectives

Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. The aim of this study was to characterize apelin and APJ expression in human osteoblasts and to investigate the effects of apelin on osteoblasts.

Results

Apelin and APJ were expressed in human osteoblasts. Apelin stimulated proliferation of human osteoblasts, but had no effect on alkaline phosphatase (ALP) activity, osteocalcin and type I collagen production in human osteoblasts. Suppression of APJ with small-interfering RNA (siRNA) abolished the apelin-induced cell proliferation. Apelin induced activation of Akt (Phosphatidylinositol-3 kinase downstream effector), but not MAPKs, such as c-jun N-terminal Kinase (JNK), p38 and ERK1/2 in human osteoblasts. This effect was blocked by suppression of APJ with siRNA. Furthermore, LY294002 (PI3 kinase inhibitor) blocked the activation of Akt by apelin and abolished the apelin-induced cell proliferation.

Conclusions

Human osteoblasts express apelin and APJ and apelin enhances human osteoblast proliferation, but has no effect on osteoblast differentiation, and APJ/PI3 kinase/Akt pathway is involved in the proliferation response. These findings suggest that apelin may function as a mitogenic agent for 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).

References (53)

  • M. Hosoya et al.

    Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin

    J Biol Chem

    (2000)
  • M. Jaszberenyi et al.

    Behavioral, neuroendocrine and thermoregulatory actions of apelin-13

    Neuroscience

    (2004)
  • D. Sunter et al.

    Intracerebroventricular injection of apelin-13 reduces food intake in the rat

    Neurosci Lett

    (2003)
  • M. Sorhede Winzell et al.

    The apj receptor is expressed in pancreatic islets and its ligand, apelin, inhibits insulin secretion in mice

    Regul Pept

    (2005)
  • A. Kasai et al.

    Apelin is a novel angiogenic factor in retinal endothelial cells

    Biochem Biophys Res Commun

    (2004)
  • K.J. Stewart et al.

    Fitness, fatness and activity as predictors of bone mineral density in older persons

    J Intern Med

    (2002)
  • R. Lindsay et al.

    Bone mass and body composition in normal women

    J Bone Miner Res

    (1992)
  • D.T. Felson et al.

    Effects of weight, and body mass index on bone mineral density in men and women

    J Bone Miner Res

    (1993)
  • H.S. Glauber et al.

    Body weight versus body fat distribution, adiposity, and frame size as predictors of bone density

    J Clin Endocrinol Metab

    (1995)
  • S. Khosla et al.

    Relationship between body composition and bone mass in women

    J Bone Miner Res

    (1996)
  • N. De Mota et al.

    Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release

    Proc Natl Acad Sci U S A

    (2004)
  • L. Wei et al.

    Regulation of apelin mRNA expression by insulin and glucocorticoids in mouse 3T3-L1 adipocytes

    Regul Pept

    (2005)
  • J. Boucher et al.

    Apelin, a newly identified adipokine up-regulated by insulin and obesity

    Endocrinology

    (2005)
  • D.K. Lee et al.

    Characterization of apelin, the ligand for the APJ receptor

    J Neurochem

    (2000)
  • A.D. Medhurst et al.

    Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin

    J Neurochem

    (2003)
  • B. Masri et al.

    Apelin (65–77) activates p70 S6 kinase and is mitogenic for umbilical endothelial cells

    Faseb J

    (2004)
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