Elsevier

Biochemical Pharmacology

Volume 61, Issue 3, 1 February 2001, Pages 319-326
Biochemical Pharmacology

Molecular and cellular pharmacology
Signal transduction system for interleukin-6 and interleukin-11 synthesis stimulated by epinephrine in human osteoblasts and human osteogenic sarcoma cells

https://doi.org/10.1016/S0006-2952(00)00544-XGet rights and content

Abstract

1Epinephrine increased gene- and protein-expression of interleukin-6 (IL-6) and interleukin-11 (IL-11), which are capable of stimulating the development of osteoclasts from their hematopoietic precursors, in human osteoblast (SaM-1) and human osteosarcoma (SaOS-2, HOS, and MG-63) cell lines. An increase in IL-6 and IL-11 synthesis in response to epinephrine appeared to be a common feature in osteoblastic cells, but the magnitude of expression was different in these cell lines. In HOS cells treated with epinephrine, increases of IL-6 and IL-11 synthesis were inhibited by timolol (a β-blocker), H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; an inhibitor of protein kinase A (PKA)) and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; an inhibitor of p38 mitogen-activated protein kinase (MAPK)], but not by phentolamine (an α-blocker), calphostin C [an inhibitor of protein kinase C (PKC)], or PD98059 (2′-amino-3′-methoxyflavone; an inhibitor of classic MAPK), suggesting a common pathway mediated by β-adrenergic receptors in the PKA and p38 systems involved in the signal transduction of IL-6 and IL-11. Furthermore, expression of both genes was inhibited by curcumin [an inhibitor of activating protein-1 (AP-1) activation], but not by pyrrolidine dithiocarbamate (PDTC) [an inhibitor of nuclear factor (NF)-κB]. The pharmacological study suggested that coinduction of the two genes in response to epinephrine occurred via activation of AP-1. The findings of the present study suggest that coinduction of IL-6 and IL-11 in response to epinephrine probably occurs via the PKA and p38 MAPK systems, leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Introduction

It is well known that osteoblasts and marrow stromal cells are capable of producing a wide array of factors that can potentially act as autocrine and paracrine regulators of bone cell function. Several cytokines that can be produced by stromal cells and osteoblasts, such as colony-stimulating factors, IL-6, and IL-11, may affect osteoclast recruitment rather than the function of mature cells [1]. The synthesis of IL-6 and IL-11, which are capable of stimulating the differentiation of osteoclasts from their hematopoietic precursors [2], [3], is stimulated by various systemic and local factors, such as parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), tumor necrosis factor (TNF), transforming growth factor (TGF), and IL-1 [4], [5], [6].

The signal transduction systems involved in these hormonal actions on IL-6 and IL-11 synthesis have been drawing increasing attention to aid in understanding the intracellular network of proteins that transduce extracellular signals to intracellular responses in osteoblasts. Recent studies reported that the PKA pathway is involved in prostaglandin E2 (PGE2)-induced IL-6 synthesis [7], and in PTH-induced IL-6 and IL-11 synthesis [6]; the PKC pathway is involved in basic fibroblast growth factor- and PGF-induced IL-6 synthesis [8], [9] and IL-1-induced IL-6 and IL-11 synthesis [6]. In addition, MAPKs are important mediators of intracellular signaling; an inhibitor for MAPKs has been reported to inhibit sphingosine 1-phosphate- and IL-1β-induced IL-6 synthesis [10], [11].

Osteoblasts have been demonstrated to express β-adrenergic and cytokine receptors [12]. Stimulation of bone resorption by adrenergic agonists and an anabolic action on bone by the β-adrenergic antagonist have been demonstrated. In neonatal mouse calvaria, an adrenergic receptor agonist increased cAMP production and stimulated bone resorption [13]. Propranolol inhibited cAMP formation induced by β-adrenergic receptor agonists in bone organ culture [14] and increased bone strength and the rates of endochondral bone formation in rats [15]. Stimulation of β-adrenergic receptors in rat osteoblast-like osteosarcoma cells (UMR106–01) has been demonstrated to enhance the cAMP signaling pathway, which regulates IL-6 synthesis [16], leading to increased osteoclastic bone resorption.

To date, the signal transduction systems involved in stimulating IL-6 and IL-11 synthesis by epinephrine have not been studied in human osteoblasts. In the present study, we characterized the signal transduction systems involved in the stimulation of IL-6 and IL-11 production in human osteoblasts and in human osteosarcoma cell lines, using RT–PCR and the ELISA method. In addition, we investigated the transcriptional mechanisms regulating the expression of IL-6 and IL-11, using an inhibitor of NF-κB and AP-1 activation.

Section snippets

Materials

SaM-1 cells were prepared from an explant of ulnar periosteum from a 20-year-old male patient undergoing curative surgery, with informed consent. These cells have a mitotic life span of 34 population doubling levels (PDLs) [17]. SaM-1 cells were used at 23–24 PDLs in our experiments. SaOS-2 cells were obtained from the RIKEN Cell Bank, and HOS and MG-63 cells were obtained from the American Type Culture Collection. α-MEM was purchased from Gibco BRL, and FBS from Cell Culture Laboratories and

Stimulation by epinephrine of the production of IL-6 in osteoblasts

Figure 1 shows the effect of epinephrine on IL-6 production in SaM-1 cells. Cells were treated with the indicated concentration of epinephrine for the indicated periods, and the conditioned medium was analyzed for IL-6 production using an ELISA system. IL-6 production was increased by epinephrine in a time- and concentration-dependent manner in the conditioned medium of SaM-1 cells (Fig. 1). The induction of IL-6 production was rapid (as early as 1 hr after treatment), and the effect of

Discussion

In the present study, we demonstrated the coinduction of IL-6 and IL-11 in human osteoblasts (SaM-1) and human osteosarcoma cells (SaOS-2, HOS, and MG-63). The increase in IL-6 and IL-11 synthesis in response to epinephrine appears to be a common feature in osteoblastic cells, but the magnitude of expression was different in these cell lines. IL-6 is a potent regulator for osteoclast differentiation and elicits bone resorption inin vivo andin vitro models that contain early osteoclast

Acknowledgments

This study was supported, in part, by a grant-in-aid for Scientific Frontier Promoted Research and by a grant-in-aid from the Ministry of Education, Science, Sport and Culture of Japan (No. 11671861 to A.T.).

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