Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability

doi:10.1016/S0006-2952(03)00450-7

Biochemical Pharmacology

Volume 66, Issue 9, 1 November 2003, Pages 1809-1819

https://doi.org/10.1016/S0006-2952(03)00450-7 Get rights and content

Abstract

In bone biology, interleukin (IL)-6 is an autocrine/paracrine cytokine which can induce osteoclasts formation and activation to help mediate inflammatory bone destruction. Previous studies have shown that tetracycline and its derivatives have potentially beneficial therapeutic effects in the prevention and treatment of metabolic bone diseases by modulating osteoblast and osteoclast activities. Our previous studies indicated that non-antimicrobial chemically modified tetracyclines (CMTs) can dose-dependently inhibit IL-1β-induced IL-6 secretion in osteoblastic cells. In the present study, we explored the molecular mechanisms underlying the ability of doxycycline analogs CMT-8 and its non-chelating pyrazole derivative, CMT-5 to affect IL-6 gene expression in murine osteoblasts. Steady-state IL-6 mRNA was decreased with CMT-8 (ca. 50%) but not by CMT-5 when stimulated by IL-1β. CMT-8 regulation of IL-1β-induced IL-6 gene expression was further explored. CMT-8 did not affect IL-6 promoter activity in reporter gene assays. However, the IL-6 mRNA stability was decreased in the presence of CMT-8. These effects require de novo protein synthesis as they were inhibited by cycloheximide. Western blot analysis indicated that CMT-8 did not affect p38 mitogen-activated protein kinase, c-jun NH₂-terminal kinases, or extracellular signal-regulated kinases (1 and 2) phosphorylation in response to IL-1β. These data suggest that CMT-8 can modulate inhibit IL-1β-induced IL-6 expression in MC3T3-E1 cells at the post-transcriptional level affecting IL-6 mRNA stability. These observations may offer a novel molecular basis for this treatment of metabolic bone diseases that are mediated by IL-6.

Introduction

Bone is a dynamic tissue that constantly undergoes a remodeling process where bone resorption and bone deposition are balanced. When chronic inflammation occurs in bone, this balance is disrupted favoring net bone loss. Diseases involving an intimate combination of bone loss and inflammation include rheumatoid arthritis (RA) periodontal disease, and other metabolic bone diseases. Inflammatory cytokines, such as IL-1α and IL-1β, tumor necrosis factor (TNF)-α [1], [2], and IL-17 [3] have all been shown to promote tissue destruction. In addition to these factors, members of the IL-6 family of cytokines have been shown to perturb bone and cartilage metabolism [4]. For example, leukemia inhibitory factor (LIF) and oncostatin M (OSM) have been shown to promote cartilage degradation in vitro and IL-6, LIF [5], OSM [6], IL-11 [7] have all been shown to mediate tissue destruction in various experimental models of bone resorption.

IL-6 is a pleiotropic cytokine that elicits a wide variety of immune/inflammatory responses including B- and T-cell activation, stimulation of fever, and release of acute phase response proteins [8]. Bone resorptive agents, such as parathyroid hormone (PTH), TNF-α, and IL-1β, have all been shown to stimulate IL-6 production and secretion in osteoblasts [9], [10]. Proinflammatory cytokines, including those of the IL-6 family, involved in bone formation and remodeling, converge on the expression of receptor-activated NF-κB ligand (RANKL), and its decoy receptor, osteoprotegerin (OPG) [11], [12]. RANKL and OPG expression from stromal/osteoblastic cells increases osteoclastogenesis through activation of the RANKL cognate receptor, RANK, located on osteoclast precursor cells [13], [14]. Thus, inhibition of IL-6 expression presents an attractive target for the treatment of metabolic bone diseases.

Tetracycline and its analogs have been shown to inhibit bone resorption in organ culture when induced by PTH, prostaglandin E₂ (PGE₂), or bacterial lipopolysacchride (LPS) [15], [16], [17]. Other studies have shown that tetracyclines inhibit excessive connective tissue degradation in several pathological conditions, including periodontitis and streptozotocin-induced diabetes [15], [18]. Tetracyclines are potent inhibitors of extracellular matrix metalloproteinases (MMPs), including the three collagenases MMP-1, MMP-8, and MMP-13, and two gelatinases MMP-2 and MMP-9 [19], [20], [21], [22], [23]. More recently, tetracyclines and their analogs have been shown to inhibit inflammatory mediators, such as nitric oxide synthase [24], [25], PGE₂ production [26], and possibly other cytokines [27]. Our group has also presented evidence that cytokine-induced IL-6 secretion is regulated by CMTs [28].

Since tetracycline is well known for its ability to bind divalent cations, such as calcium and zinc, and affect intracellular calcium concentrations, we evaluated the abilities of CMTs, which lack antimicrobial activity to affect osteoblast IL-6 secretion from MC3T3-E1 osteoblast cells. Previous data from our laboratory had indicated that regulation of intracellular calcium stores were critical to stimulus-secretion coupling in osteoblasts [29], [30], [31]. Herein, we show that, CMT-8 and but not CMT-5, an imidazole derivative, can decrease IL-6 gene expression when induced by IL-1β by a mechanism that requires de novo protein synthesis in a manner that decreases IL-6 mRNA stability.

Section snippets

Materials

CMTs were obtained from CollaGenex, Inc., under a material transfer agreement between SUNY at Buffalo and CollaGenex, Inc. Human IL-1β was obtained from Sigma.

Cell culture of MC3T3-E1 cells

MC3T3-E1 osteoblastic cells were obtained from RIKEN. Cells were cultured in DMEM (Invitrogen Life Technologies), supplemented with 10% fetal bovine serum (Sigma), 100 units/mL penicillin, and 100 μg/mL streptomycin, in a humidified atmosphere of 5% CO₂ in air at 37°. Osteoblast phenotypic mRNAs, including bone sialoprotein and osteocalcin,

CMT-8, but not CMT-5, decreases steady-state IL-6 mRNA levels in MC3T3-E1 cells and primary rat osteoblastic cells

Northern blot analysis and semi-quantitative RT–PCR analysis were used to determine CMT-8 and -5 effects on IL-1β-induced IL-6 expression in MC3T3-E1 cells. IL-6 mRNA species of ∼2.4 kb was detected in MC3T3-E1 cells by Northern blot analysis (Fig. 1A). RT–PCR analysis of IL-6 reproducibly identified a 638 bp fragment which was authenticated by sequencing as IL-6 gene product (Fig. 1B). As shown previously in these cells, IL-1β increases steady-state mRNA levels [9], [34]. When cells are

Discussion

Tetracyclines have been used for decades as antimicrobial agents. More recently, a new effect of this class of agents has been discovered: inhibition of MMPs, which degrade connective tissue and other host-modulating activities [16], [41]. The effects of tetracyclines were found to be independent of their antimicrobial activity in germ-free and conventional animal models, as well as in sterile in vitro systems [20]. Additionally, some CMTs, which lack antimicrobial activity, inhibit

Acknowledgements

This work was supported, in part through the Moir P. Tanner Research Fund (K.L.K.), the ADA Health Foundation (K.L.K.), and CollaGenex, Inc. (K.L.K.).

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Chemically modified tetracyclines selectively inhibit IL-6 expression in osteoblasts by decreasing mRNA stability

Abstract

Introduction

Section snippets

Materials

Cell culture of MC3T3-E1 cells

CMT-8, but not CMT-5, decreases steady-state IL-6 mRNA levels in MC3T3-E1 cells and primary rat osteoblastic cells

Discussion

Acknowledgements

Baillieres Clin. Rheumatol.

Bone

Rheum Dis. Clin. North Am.

Arch. Oral Biol.

Bone

J. Biol. Chem.

J. Vasc. Surg.

Bone

Biochem. Biophys. Res. Commun.

FEBS Lett.

Int. Immunopharmacol.

Mol. Cell. Biol. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Bone

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Genet. Dev.

J. Biol. Chem.

J. Biol. Chem.

Immunopharmacology

Pharmacol. Ther.

Bone

Cytokines in rheumatoid arthritis

Curr. Rheumatol. Rep.

Synovial cells from a patient with rheumatoid arthritis produce osteoclastogenesis inhibitory factor/osteoprotegerin: reciprocal regulation of the production by inflammatory cytokines and basic fibroblast growth factor

J. Bone Miner. Metab.

Leukaemia inhibitory factor stimulates proteoglycan resorption in porcine articular cartilage

Rheumatol. Int.

Role of cytokines in bone resorption

Bone

Interleukin-6 messenger RNA expression and interleukin-6 protein secretion in cells isolated from normal human bone: regulation by interleukin-1

J. Bone Miner. Res.

Stimulation of interleukin-6 promoter by parathyroid hormone, tumor necrosis factor alpha, and interleukin-1beta in UMR-106 osteoblastic cells is inhibited by protein kinase C antagonists

J. Bone Miner. Res.