Adenyl Cyclase and Interleukin 6 Are Downstream Effectors of Parathyroid Hormone Resulting in Stimulation of Bone Resorption Edward M. Greenfield, Steven M. Shaw, Sandra A. Gomik, and Michael A. Banks Department of Orthopaedics and Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106-5000 Abstract * osteoclast * cytokine * cell-cell interactions * bone resorp- tion Parathyroid hormone and other bone resorptive agents function, at least in part, by inducing osteoblasts to secrete Introduction cytokines that stimulate both differentiation and resorptive activity of osteoclasts. We previously identified two poten- Net bone loss, in conditions such as osteoporosis, results from tially important cytokines by demonstrating that parathy- a disturbance in the normal balance between bone resorption roid hormone induces expression by osteoblasts of 1L-6 and by osteoclasts and bone formation by osteoblasts. Thus, knowl- leukemia inhibitory factor without affecting levels of 14 edge of the mechanisms that regulate both resorption and forma- other cytokines. Although parathyroid hormone activates tion of bone is fundamental to the understanding of the patho- multiple signal transduction pathways, induction of IL-6 genesis of these diseases. and leukemia inhibitory factor is dependent on activation In addition to forming bone, osteoblasts regulate osteoclast of adenyl cyclase. This study demonstrates that adenyl cy- resorptive activity in response to parathyroid hormone (PTH) clase is also required for stimulation of osteoclast activity and other resorptive agents (1, 2). This concept is best sup- in cultures containing osteoclasts from rat long bones and ported by evidence that many agents, including PTH, only stim- UMR106-01 rat osteoblast-like osteosarcoma cells. Since ulate osteoclast resorptive activity in the presence of osteoblasts the stimulation by parathyroid hormone of both cytokine or conditioned media from osteoblasts that had been exposed production and bone resorption depends on the same signal to PTH (3, 4). The primary effect of PTH is therefore believed transduction pathway, we hypothesized that IL-6 might be to be stimulation of production by osteoblasts of soluble cyto- a downstream effector of parathyroid hormone. We found kines that, in turn, function as downstream effectors to activate that addition of exogenous IL-6 mimics the ability of para- osteoclasts. However, the identity and functions of these critical thyroid hormone to stimulate bone resorption. More im- cytokines are unknown. portantly, an antibody directed against the IL-6 receptor To identify osteoblast-derived cytokines that may be im- blocks moderate stimulation of osteoclast activity induced portant in stimulation of bone resorption, we previously exam- by the hormone. Interestingly, strong stimulation of resorp- ined the effect of PTH on expression by osteoblasts of 16 differ- tion overcomes this dependence on IL-6. Thus, parathyroid ent cytokine genes (5). The only mRNAs that were responsive hormone likely induces multiple, redundant cytokines that to PTH were those encoding interleukin 6 (IL-6) and leukemia can overcome the 1L-6 requirement associated with moder- inhibitory factor (LWF),1 which were rapidly and transiently ate stimulation. Taken together with studies showing that stimulated. Maximal stimulation was - 10-50-fold. We and many other bone resorptive agents also stimulate 11-6 pro- others have also found that secretion of both IL-6 and LWF duction, our results suggest that 1L-6 may be a downstream proteins is stimulated by PTH (5-1 1 ) as well as by many other effector of these agents as well as of parathyroid hormone. resorptive agents, including PTH-related protein, IL-1, tumor (J. Clin. Invest. 1995. 96:1238-1244.) Key words: osteoblast necrosis factor, 1,25-dihydroxyvitamin D3, transforming growth factor-f, vasoactive intestinal peptide, isoproterenol, and lipo- polysaccharide (5, 6, 8-10, 12-16).2 Demonstration that PTH Portions of this work have been presented in preliminary form at annual and other resorptive agents induce IL-6 production by osteo- meetings of the American Geriatrics Society in New Orleans, LA on blasts in conjunction with reports that IL-6 stimulates bone 15-19 November 1993, the American Society for Bone and Mineral resorption (7, 9, 12) suggests that osteoblast-derived 1L-6 might Research in Tampa, FL on 18-22 September 1993 and in Kansas City, be a common downstream effector of many bone resorptive MO on 9-13 September 1994, the American Society for Cell Biology agents (5). Although in particular cell preparations PITH does in New Orleans, LA on 11-15 December 1993, the Arthritis Foundation in Phoenix, AZ on 18-20 June 1993, and the Orthopaedic Research not stimulate IL-6 production (9, 10, 12) and IL-6 does not Society in Washington, DC on 17-20 February 1992 and in San Fran- induce resorption (17-19), these discrepancies are likely due cisco, CA on 15-18 February 1993. to investigation of cells at different developmental stages or Address correspondence to Edward M. Greenfield, Ph.D., Depart- lack of adequate assay sensitivity (for review see reference 5). ment of Orthopaedics, Case Western Reserve University, 11100 Euclid The importance of IL-6 is underscored by recent findings that Avenue, Cleveland, OH 44106-5000. Phone: 216-368-1331; FAX: 216- implicate this cytokine in the increased bone resorption that 368-1332. occurs during estrogen withdrawal (2, 14, 20, 21), in Paget's Received for publication 30 January 1995 and accepted in revised form 23 May 1995. J. Clin. Invest. 1. Abbreviations used in this paper: LWF, leukemia inhibitory factor; X The American Society for Clinical Investigation, Inc. TRAP, tartrate-resistant acid phosphatase. 0021-9738/95/09/1238/07 $2.00 2. Greenfield, E. M., M. C. Horowitz, and S. A. Gormik, manuscript Volume 96, September 1995, 1238-1244 submitted for publication. 1238 Greenfield, Shaw, Gornik, and Banks disease (22, 23), in Gorham-Stout disease (24), and in some 1.0 Figure 1. PTH-(3-34) is forms of hypercalcemia of malignancy (25). unable to mimic the abil- 0.8 PTH activates both the classical adenyl cyclase signal trans- Z ity of PTH-(1-34) to 1 34 stimulate resorption. Os- duction pathway as well as the phospholipase C pathway 2L IC teoblast/osteoclast co- through binding to a single receptor (26). Pharmacological acti- 0.4 W - - 3 4 cultures were treated vation of either pathway can stimulate bone resorption (27- w1 z26i3 020 4- with the indicated con- 29). To fully understand the mechanism by which PTH stimu- centrations of PTH- (1- lates bone resorption, it is therefore important to clarify which 0.0 N 34), PTH-(3-34), or pathway(s) is involved in this process. PTH-(3-34), a partial 0 .1 1 10 100 acetic acid vehicle (final agonist that activates phospholipase C but not adenyl cyclase, HORMONE (nM) concentration = 10 jtM) has proven useful to determine whether adenyl cyclase is in- for 24 h. Data are presented as the area resorbed (percentage of the volved in a particular response (30, 31). Previously, we used total bone slice area) ±SEM (n = 4). *P = 0.043, **P = 0.004, ***P PTH-(3-34) to demonstrate that adenyl cyclase activation is = 0.0005. required for induction of IL-6 mRNA and protein in osteoblasts by PTH.2 In this study, we demonstrate that activation of adenyl cyclase is also required for stimulation of bone resorption. These debris to settle out, the cell suspension was transferred to a 35-mm Petri results led us to examine whether LL-6 is one of the crucial dish containing 30 slices (3 mm x 3 mm x 0.2 mm) of bovine cortical cytokines produced by osteoblasts to stimulate osteoclast re- bone or sperm whale dentin (kindly provided by Dr. C. Potter, National sorptive activity. We show that addition of exogenous LL-6 can Museum of Natural History, Washington, DC). After incubation for 15 mimic the effect of PTH on bone resorption and that moderate min (5% CO2, 370C), bone slices were rinsed in PBS and transferred stimulation by PTH requires IL-6 production. Interestingly, this in groups to wells (2 cm2) containing preincubated media (lacking on can be overcome stimulation of Hepes). UMR106-01 cells were added at a density of 50,000/well to dependence IL-6 by strong ensure PTH responsiveness. After 2 h (5% CO2, 370C), potential resorp- osteoclast activity by PTH. tive agents were added and the cultures (total volume = 0.4 ml) were incubated for another 24 h. Bone and dentin slices were sonicated in Methods Triton X-100, rinsed in PBS, fixed in glutaraldehyde, dehydrated in ethanol, and stained with toluidine blue as described (35). Resorption lacunae were measured in blinded fashion by computer-assisted histo- were obtained from Bachem California PTH-(1-34) and PTH-(3-34) morphometry (Bioquant System IV; R&M Biometrics, Inc., Nashville, (Torrance, CA); and recombinant murine IL-6 was from Genzyme TN). Total resorbed areas are reported as a percentage of the total Corp. (Boston, MA). All of these agents were screened for endotoxin surface area of the slice ±SEM. Experiments were excluded from further contamination using the colorimetric Limulus amebocyte lysate assay analysis if stimulated cultures exhibited SEM > 20% of the mean. (No. QCL-1000; Whittaker M. A. Bioproducts, Walkersville, MD). For All reported data are representative of multiple experiments, with the the hormones, endotoxin levels were < 0.0008 Eu/ml for the highest exception of Fig. 5 which is a compendium of six experiments. concentration used in the experiments and were < 0.1 Eu/ml for the Osteoclast number was determined in some by staining highest concentration of IL-6 used. Moreover, none of these agents experiments the bone or dentin slices for tartrate-resistant acid phosphatase (TRAP) revealed significant endotoxin contamination in our previously described using a commercial kit (No. 387-A; Sigma Immunochemicals), but (5) functional assay expression in osteoblastic examining cytokine increasing the tartrate concentration to 50 mM.