- Email: [email protected]
Effect of phosphodiesterase inhibitor-4, rolipram, on new bone formations by recombinant human bone morphogenetic protein-2
Bone Vol. 30, No. 4 April 2002:589 –593
Effect of Phosphodiesterase Inhibitor-4, Rolipram, on New Bone Formations by Recombinant Human Bone Morphogenetic Protein-2 H. HORIUCHI, N. SAITO, T. KINOSHITA, S. WAKABAYASHI, N. YOTSUMOTO, and K. TAKAOKA Department of Orthopaedic Surgery, Shinshu University School of Medicine, Nagano, Japan
terials in combination with growth factors such as bone morphogenetic proteins (BMPs), which exhibit a strong bone-inducing capacity. Currently, some of the human BMPs can be produced successfully by DNA recombination technology and have been combined with collagenous carriers for use in the field of orthopedic and craniofacial surgery. However, ongoing challenges have limited the routine use of these bone graft substitutes. Low responsiveness to BMP in humans as compared with rodents and the consequent requirement of a high dose of BMP are some of the problems.2– 4,7,11,14 Experimental data have shown that milligram quantities of rhBMP-2 are required to elicit ectopic new bone in both primates and humans (data not shown). Therefore, an improvement in the responsiveness to BMP or performance of BMP itself is an important issue for its effective and economical use in the clinical setting. We have recently demonstrated that long-term administration of a general phosphodiesterase inhibitor, Pentoxifylline (PTX), and the phosphodiesterase-4 (PDE-4)-selective inhibitor, Rolipram, could increase bone mass in normal mice by enhancing physiological bone formation.13 This was most likely achieved by increasing cyclic nucleotide (cAMP) levels in osteogenic cells. The PDEs are a large group of structurally related isoenzymes derived from at least 11 distinct genes. They can be divided into 11 groups on the basis of their substrate specificity, selective inhibition or stimulation by cofactors, selective inhibition by standard inhibitors, and gene homology.15 Based on our previous results and a recent report from another group describing PDE-4 isomer predominantly distributed in osteoblasts,1 we examined the effect of Rolipram on BMP-dependent bone formation as one approach to improve the performance of BMP in this osteoinductive protein.
Collagen sponge disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 g/disk) and implanted onto the back muscles of mice. Ten or 20 mg/kg per day of Rolipram, a selective inhibitory agent to phosphodiesterase type 4 (PDE-4), or vehicle, was injected subcutaneously into the host mice for 3 weeks. After treatment, rhBMP-2-induced ectopic ossicles were harvested and examined by radiographic and histologic methods to determine the size, bone quality, and mineral content of the ossicles. The ossicles from a group treated with 20 mg/kg per day Rolipram were significantly larger in size and higher in bone mineral density (BMD) and bone mineral content (BMC) than the control samples. No significant differences were noted in mice treated with 10 mg/kg per day of Rolipram. Histologically, ossicles from the high-dose (20 mg/kg per day) Rolipram-treated group showed densely packed, thicker trabeculae when compared with those from the control group. These experimental results indicate that the PDE-4 inhibitor, Rolipram, may enhance the bone-inducing capacity of BMP-2 in mesenchymal cells. This in turn may result in increased responsiveness to BMP-2 and point to a potential use of PDE-4 inhibitors for the promotion of rhBMP-dependent bone repair. (Bone 30: 589 –593; 2002) © 2002 by Elsevier Science Inc. All rights reserved. Key Words: Bone morphogenetic protein (BMP); Ectopic bone formation; Rolipram; Phosphodiesterase-4 (PDE-4) inhibitor. Introduction
Materials and Methods
Fracture and the regenerative repair of bone defects associated with tumor surgery are often impaired due to inadequate fixation, extensive soft tissue damage, and the size of the bone defect and/or infection. For treatment of these types of cases, autogenous (vascularized or free graft) bone grafting has been routinely indicated despite significant disadvantages such as functional and cosmetic morbidity at the donor site and a limited source of donor bone. One of the most promising approaches for overcoming the disadvantages of autografting involves the use of bioma-
Preparation of BMP-containing Collagen Pellets rhBMP-2 was produced by the Genetics Institute (Cambridge, MA) and donated to us through Yamanouchi Phamaceutical Co. (Tokyo, Japan). The rhBMP-2 was provided in a buffer solution (5 mmol/L glutamic acid, 2.5% glycine, 0.5% sucrose, and 0.01% Tween-80) at a concentration of 1 G/L after filter sterilization. To prepare one implant sample, 5 L (5 g of rhBMP) of the rhBMP-2 solution was added to 20 L of 0.01N HCl solution and blotted onto a collagen sponge disk (6 mm diameter, 1 mm thickness) fabricated from commercially available bovine collagen sheets (Helistat, Integra Life Sciences Corp., Plainsboro, NJ), freeze-dried, and kept at ⫺20°C until
Address for correspondence and reprints: Dr. Hiroshi Horiuchi, Department of Orthodpaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, Japan. E-mail: [email protected]. shinshu-u.ac.jp © 2002 by Elsevier Science Inc. All rights reserved.
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implantation into mice. All procedures were carried out under sterile conditions. Rolipram Rolipram was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in physiological saline prior to use. Experimental Protocols Thirty male ddy mice, 4 weeks of age, were purchased from Nippon SLC Co. (Shizuoka, Japan) and housed in cages with free access to food and water for 1 week. Prior to surgery to implant the collagen sponge disks with rhBMP-2, the mice were anesthetized with diethylether. The pellets were implanted into the left dorsal muscle pouches (one pellet per animal) and divided into three groups depending on the dose of Rolipram. Zero (vehicle alone), 10 mg/kg body weight (BW)/day (low-dose Rolipram; LR), or 20 mg/kg BW/day (high-dose Rolipram; HR), of Rolipram was injected subcutaneously for 3 weeks. At the end of this period, the implants were harvested to evaluate size, bone mineral density (BMD), and bone mineral content (BMC) of the respective BMP-induced ossicle. The volume of the each ossicle was determined based on three mutually orthogonal measurements (a, b, and c) of the ossicle. These measurements were made using a caliper. The formula, V ⫽ abc/6, was used to compute ossicle volumes.7,10 All harvested tissues were radiophotographed with a soft X-ray apparatus (Sofron Co., Ltd., Tokyo, Japan). To quantitate bone quality and mass of the ossicles, BMD (milligrams per square centimeter) and BMC (milligrams) of each ossicle were measured by single-energy X-ray absorptiometry (SXA) using a bone mineral analyzer (DCS-600R, Aloka Co., Tokyo). The harvested tissues from each group were then fixed in neutralized 10% formalin, defatted in chloroform, decalcified with 10% ethylenediamine tetraacetic acid (EDTA), and embedded in paraffin wax. Sections of 5 m in thickness were cut, stained with hematoxylin-eosin, and examined under a light microscope. At 0 (control), 1, 2, and 3 weeks after implantation, blood was collected from the Rolipram-treated mice (20 mg/kg per day) and stored at ⫺20°C until biochemical measurements were performed. Serum osteocalcin was measured by immunoradiometric assay (IRMA) using a commercial kit (Immutopics, Inc., San Clemente, CA). All procedures for the animal experiments were carried out in compliance with the guidelines of the institutional animal care committee of Shinshu University.
Figure 1. Volume of harvested tissues. Volume level in the Rolipram 20 mg/kg per day group (HR) was significantly larger than in the control group (n ⫽ 10). Data expressed as mean ⫾ SD. **Significantly different from controls (p ⬍ 0.01).
significant difference vs. controls was found in the LR group (65.1 ⫾ 8.8 mm3). Radiographic Findings Areas of calcified mass on the radiograms from the HR group were significantly larger than those observed in the control group. The trabeculae in the ossicles of the HR group were more densely packed than those in the control ossicles (Figure 2). Bone Mineral Density of Ossicles Figure 3 shows the mean BMD of the ossicles from each of the groups. There was no significant difference in BMD between the LR group (15.9 ⫾ 3.6 mg/cm2) and the control group (14.5 ⫾ 2.5 mg/cm2). However, the BMD of the HR group (21.0 ⫾ 3.5 mg/cm2) was significantly higher than that of the control group.
Statistical Analysis Quantitative data were expressed as the mean ⫾ SD. Differences between experimental groups were considered statistically significant at p ⬍ 0.05 using Student’s t-test. Results Mass of rhBMP-2-induced Ossicles Figure 1 shows the mean mass of ossicles from the three groups at 3 weeks after implantation. The mean harvested bone mass of the control group was 53.1 ⫾ 7.5 mm3. The bone mass from the high-dose Rolipram (HR) group was 82.6 ⫾ 9.0 mm3 and significantly higher than that found in the control group (p ⬍ 0.01). The daily injection of a high dose of Rolipram (20 mg/kg per day) resulted in the formation of ectopic ossicles 1.5 times larger in size and with densely packed trabeculae. However, no
Figure 2. Soft X-ray photograph of the ossicle formed at 3 weeks after implantation. A typical implant from each group is shown. (A) Control group. (B) Rolipram 10 mg/kg per day group. (C) Rolipram 20 mg/kg per day group. The radio-opaque areas of the Rolipram 10 and 20 mg/kg per day groups are larger than that of the control group. Also, the radioopaque density of the harvested tissue from the Rolipram 10 and 20 mg/kg per day groups is higher than that of the control group. Scale bar ⫽ 6 mm.
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Figure 3. Effect of Rolipram treatment on bone mineral density (BMD). BMD from harvested tissues of the 20 mg/kg per day Rolipram group was significant higher than that of the control group (n ⫽ 10). Data presented as mean ⫾ SD. *Significantly different from controls (p ⬍ 0.05).
Bone Mineral Content of Ossicles Mean BMC values of the ossicles from each group are shown in Figure 4. The mean BMC of ossicles from the HR group (15.7 ⫾ 4.7 mg) was significantly greater than that of the control group (8.4 ⫾ 2.4 mg, p ⬍ 0.01). The BMC in the LR group (11.2 ⫾ 3.3 mg) revealed an upward trend but no significant difference when compared with the control group.
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Figure 5. Photomicrographs of the harvested tissue at 3 weeks after implantation. (hematoxylin-eosin stain; original magnification ⫻100). (A) Control. (B) Rolipram 10 mg/kg per day. (C) Rolipram 20 mg/kg per day. New bone formation with hematopoietic marrow and bony trabeculae is visible in the tissue. In the 20 mg Rolipram-treated group, there are visible increases in number and thickness and quantity of bony trabeculae when compared with the control group.
weeks of daily Rolipram treatment. However, serum osteocalcin increased significantly after 3 weeks of treatment (117.7 ⫾ 19.7% of controls; n ⫽ 10, p ⬍ 0.01).
Histology Discussion Ossicles from all three groups revealed normal bone histology with hematopoietic marrow and bony trabeculae. In the HR group, trabeculae were increased in number and thickness when compared with the control group (Figure 5). No significant qualitative difference was noted in the LR group. Remnants of the collagen carrier were seen in the center of all the ossicles. Osteocalcin Assay Figure 6 shows the percentage changes compared with baseline values of serum osteocalcin in the Rolipram-treated mice. The increase in serum osteocalcin was not significant after 1 and 2
Figure 4. The bone mineral content (BMC) of the harvested tissues at 3 weeks after surgery. BMC was measured by single-energy X-ray absorptiometry (SXA) using a bone mineral analyzer (DCS-600R, Aloka Co., Tokyo). The BMC of the harvested tissue from the HR group was significantly higher than in the control group (p ⬍ 0.01). However, there was no significant difference in BMC between the LR group and the control group (n ⫽ 10). Data expressed as mean ⫾ SD. **Significantly different from controls (p ⬍ 0.01).
We recently reported that Pentoxifylline (PTX), a nonselective inhibitor of PDEs, could affect BMP-2-induced new bone formation in the mouse model.9 In that study, PTX treatment resulted in increases in the BMP-induced Ca content and the size of the harvested tissues. In addition, in the study presented here, Rolipram, an inhibitor of cAMP-specific PDE-4, was shown to have the capacity to enhance BMP-2-dependent ectopic new bone formation. The daily injection of a high dose of Rolipram (20 mg/kg per day) resulted in a 1.5-fold increase in the size of ectopic ossicles and twofold greater calcium content. Both nonspecific PDE inhibitor (PTX) and PDE-4-specific inhibitor (Rolipram) have been found to enhance BMP-2-dependent ectopic new bone formation. These results indicate that PDE-4 is functionally a key enzyme in the regulation of BMP action in osteoblastic differentiation, especially in the early stage. This result indicates that the PDE-4 inhibitor could be useful for promotion of BMP-dependent bone repair. Ultimately, clin-
Figure 6. Percentage changes compared with baseline values in serum osteocalcin in Rolipram-treated mice. Serum osteocalcin increased significantly after 3 weeks of Rolipram treatment (117.7 ⫾ 19.7% of controls; n ⫽ 10, p ⬍ 0.01).
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ical trials will be necessary to confirm the efficacy of this approach in humans. In an initial phase of ectopic new bone formation in the BMP-retaining collagen pellets, the pellet is resorbed and replaced by cartilage shell at the periphery of the pellet. This is then replaced by new bone through endochondral ossification. The shell determines the final size of the BMP-induced ossicle. In this study, it was determined radiographically that the calcified rings in tissues harvested from mice treated with 20 mg/kg per day of Rolipram for 1 and 2 weeks were larger than those observed in the control group (data not shown). Because the chondro-osseous differentiation of the young mesenchymal cells is initiated when these cells come into contact with BMP at the periphery of the BMP-retaining disk, this increase in bone mass may indicate an increased sensitivity and earlier initiation of the response to BMP-2. Therefore, Rolipram appears to have a stimulatory effect on the early stage of bone formation during BMP-induced osteogenesis. However, histological findings showed that new bone formations were induced during each stage of endochondral bone formation in both the control and Rolipram-treated groups (data not shown). In addition, Rolipram seemed to enhance osteoblastic function as shown by the dense distribution of thick trabeculae in the Rolipram-treated ossicles. This effect may be driven through enhancement of osteoblastic function and not by suppression of bone resorption. Earlier histomorphometric observations of bone from Rolipram-treated animals showed increased bone formation.13 We have investigated the stimulatory effects of the PDE-4 inhibitor, Rolipram, on osteoblasts lineages in vitro. This in vitro study has demonstrated that Rolipram could stimulate BMPinduced chondro-osteoblast differentiation.24 It was also shown that a selective PDE-4 inhibitor enhanced BMP-4-induced alkaline phosphatase (ALP) expression in a dose-dependent manner in osteoblastic cells.24 Finally, northern blot analysis showed that Rolipram enhanced levels of expression of the mRNAs of ALP and osteopontin in osteoblastic cells.24 These results support the findings reported herein that the PDE-4 inhibitor, Rolipram, enhances BMP-induced bone formation in vivo. The general pharmacological effects of PDE inhibitors are brought about through elevated level of intracellular cyclic nucleotides (cyclic AMP or cyclic GMP), suppressed degradation of these cyclic nucleotides, and the resulting activation of protein kinase A (PKA).1 However, little is currently known about the roles of PDE or PDE inhibitors in bone metabolism, specifically in the context of BMP-responding mesenchymal cells or osteoblasts. PDEs constitute a family of enzymes encoded by at least 20 distinct genes that can be classified into 11 groups (PDE-1 through PDE-11), depending on their enzymological characteristics.5 Further complexity can be noted because several splice variants have been identified for specific PDEs, some of which are expressed in a tissue-specific manner. The details regarding PDE isomer expression in mesenchymal or osteoblastic cells are not clear. A recent report indicated predominant distribution of cAMP-specific PDE-4 activity (about 85%) in osteoblastic cells in an vitro system.1 Previous in vivo studies conducted in our laboratory have also indicated an anabolic effect of Rolipram on normal bone formation in mice.13 The present study has also identified a significant increase in serum osteocalcin levels after 3 weeks of Rolipram treatment. Rolipram was used in this study to improve the performance of rhBMP-2. The results also indicated the apparent involvement of PDE-4 in the osteogenetic process. Splicing variants of PDE-4, which are specific to mesenchymal cells or osteoblasts, if they exist, current await identification. If a PDE-4 isomer and its
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selective inhibitory agent are identified, the agent will be utilized more effectively to promote BMP-dependent bone repair. This approach could also be used in the treatment of osteoporosis by increasing bone mass through enhanced systemic bone formation without adverse effects to other organs. With regard to the mechanism of action of Rolipram or activated PKA by elevated intracellular cAMP level in cells, two possible hypothesis must be considered: (1) suppressed production of cytokines antagonistic to BMP, such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1),6,12,16 –23,25 and consequent enhancement of BMP action; and (2) acceleration of the intracellular BMPsignaling pathway by crosstalk with the PKA-mediated signaling pathway. More precise in vitro studies are needed to test these hypotheses. Further work is underway in our laboratory to achieve this goal.
Acknowledgment: This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (No. 12671403), Japan, and grants from the Japan Rheumatism Foundation, the Hip Joint Foundation of Japan, and the Japan Orthopaedics and Traumatology Foundation, Inc. (No. 0120).
References 1. Ahlstrom, M. and Lamberg-Allardt, C. Inactivation of atrial natriuretic factorstimulated cyclic guanosine 3⬘,5⬘-monophosphate (cGMP) in UMR-106 osteoblast-like cells. Biochem Pharmacol 59:1133–1139; 2000. 2. Bostrom, M., Lane, J. M., Tomin, E., Browne, M., Berberian, W., Turek, T., Smith, J., Wozney, J., and Schildhauer, T. Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin Orthop 327:272–282; 1996. 3. Cook, S. D., Baffes, G. C., Wolfe, M. W., Sampath, T. K., Rueger, D. C., and Whitecloud, T. S., III. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Jt Surg [Am] 76:827– 838; 1994. 4. David, S. M., Gruber, H. E., Meyer, R. A., Jr., Murakami, T., Tabor, O. B., Howard, B. A., Wozney, J. M., and Hanley, E. N., Jr. Lumbar spinal fusion using recombinant human bone morphogenetic protein in the canine. A comparison of three dosages and two carriers. Spine 24:1973–1979; 1999. 5. Fawcett, L., Baxendale, R., Stacey, P., McGrouther, C., Harrow, I., Soderling, S., Hetman, J., Beavo, J. A., and Phillips, S. C. Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A. Proc Natl Acad Sci USA 97:3702–3707; 2000. 6. Hashimoto, J., Yoshikawa, H., Takaoka, K., Shimizu, N., Masuhara, K., Tsuda, T., Miyamoto, S., and Ono, K. Inhibitory effects of tumor necrosis factor alpha on fracture healing in rats. Bone 10:453– 457; 1989. 7. Heckman, J. D., Ehiler, W., Brooks, B. P., Aufdemorte, T. B., Lohmann, C. H., Morgan, T., and Botan, B. D. Bone morphogenetic protein but not transforming growth factor- enhances bone formation in canine diaphyseal nonunions implanted with a biodegradable composite polymer. J Bone J Surg [Am] 81:1717–1729; 1999. 8. Horuichi, A., Nikaido, T., Mitsushita, J., Toki, T., Konishi, I., and Fujii, S. Electroporation: A study of human uterine leiomyosarcomas in nude mice. Int J Cancer 88:640 – 644; 2000. 9. Horiuchi, H., Saito, N., Kinoshita, T., Wakabayashi, S., Tsutsumimoto, T., and Takaoka, K. Enhancement of bone morphogenetic protein-2-induced new bone formation in mice by the phosphodiesterase inhibitor Pentoxifylline. Bone 28:290 –294; 2001. 10. Hyacinthe, M., Jaroszeski, M. J., Dang, V. V., Coppola, D., Karl, R. C., Gilbert, R. A., and Heller, R. Electrically enhanced drug delivery for the treatment of soft tissue sarcoma. Cancer 85:409 – 417; 1999. 11. Itoh, H., Ebara, S., Kamimura, M., Tateiwa, Y., Kinoshita, T., Yuzawa, Y., and Takaoka, K. Experimental spinal fusion with use of recombinant human bone morphogenetic protein 2. Spine 24:1402–1405; 1999. 12. Kasyapa, C. S., Stentz, C. L., Davey, M. P., and Carr, D. W. Regulation of IL-15-stimulated TNF-alpha production by rolipram. J Immunol 163:2836 – 2843; 1999. 13. Kinoshita, T., Kobayashi, S., Ebara, S., Yoshirura, Y., Horiuchi, H., Tsutsumimoto, T., Wakabayashi, S., and Takaoka, K. Phosphodiesterase inhibitors,
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Pentoxifylline and Rolipram, increase bone mass by promoting bone formation in normal mice. Bone 27:811– 817; 2000. Lindholm, T. C., Lindholm, T. S., Alitalo, I., and Urist, M. R. Bovine bone morphogenetic protein (bBMP) induced repair of skull trephine defects in sheep. Clin Orthop 227:265–268; 1988. Manganiello, V. C., Murata, T., Taira, M., Belfrage, P., and Degerman, E. Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch Biochem Biophys 322:1–13; 1995. Nakase, T., Takaoka, K., Masuhara, K., Shimizu, K., Yoshikawa, H., and Ochi, T. Interleukin-1 beta enhances and tumor necrosis factor-alpha inhibits bone morphogenetic protein-2-induced alkaline phosphatase activity in MC3T3-E1 osteoblastic cells. Bone 21:17–21; 1997. Nyman, U., Mussener, A., Larsson, E., Lorentzen, J., and Klareskog, L. Amelioration of collagen II-induced arthritis in rats by the type IV phosphodiesterase inhibitor Rolipram. Clin Exp Immunol 108:415– 419; 1997. Pettipher, E. R., Labasi, J. M., Salter, E. D., Stam, E. J., Cheng, J. B., and Griffiths, R. J. Regulation of tumour necrosis factor production by adrenal hormones in vivo: Insights into the antiinflammatory activity of rolipram. Br J Pharmacol 117:1530 –1534; 1996. Ross, S. E., Williams, R. O., Mason, L. J., Mauri, C., Marinova-Mutafchieva, L., Malfait, A. M., Maini, R. N., and Feldmann, M. Suppression of TNF-alpha expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram. J Immunol 159:6253– 6259; 1997. Sekut, L., Yarnall, D., Stimpson, S. A., Noel, L. S., Bateman-Fite, R., Clark, R. L., Brackeen, M. F., Menius, J. A., Jr., and Connolly, K. M. Anti-
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inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation. Clin Exp Immunol 100:126 –132; 1995. Shimizu, T., Mehdi, R., Yoshimura, Y., Yoshikawa, H., Nomura, S., Miyazono, K., and Takaoka, K. Sequential expression of bone morphogenetic protein, tumor necrosis factor, and their receptors in bone-forming reaction after mouse femoral marrow ablation. Bone 23:127–133; 1998. Sounes, J., Griffin, M., Malslen, C., Ebsworth, K., Scott, L., Pollock, K., and Karlsson, M. Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF-␣ generation from human monocytes by interaction with a low-affinity phosphodiesterase 4 conformer. Br J Phamacol 118:649 – 658; 1996. Torphy, T. J. Phosphodiesterase isozymes: Molecular targets for novel antiasthma agents Am J Respir Crit Care Med 157:351–370; 1998. Wakabayashi, S., Tsutsumimoto, T., Kawasaki, S., Kinoshita, T., Horiuchi, H., and Takaoka, K. Involvement of hosphodiesterase (PDE) isozymes in osteoblastic differentiation. J Bone Miner Res 17:249 –256; 2002. Yoshikawa, H., Hashimoto, J., Masuhara, K., Takaoka, K., and Ono, K. Inhibition by tumor necrosis factor of induction of ectopic bone formation by osteosarcoma-derived bone-inducing substance. Bone 9:391–396; 1988.
Date Received: April 2, 2001 Date Revised: June 15, 2001 Date Accepted: December 19, 2001
Effect of Phosphodiesterase Inhibitor-4, Rolipram, on New Bone Formations by Recombinant Human Bone Morphogenetic Protein-2 H. HORIUCHI, N. SAITO, T. KINOSHITA, S. WAKABAYASHI, N. YOTSUMOTO, and K. TAKAOKA Department of Orthopaedic Surgery, Shinshu University School of Medicine, Nagano, Japan
terials in combination with growth factors such as bone morphogenetic proteins (BMPs), which exhibit a strong bone-inducing capacity. Currently, some of the human BMPs can be produced successfully by DNA recombination technology and have been combined with collagenous carriers for use in the field of orthopedic and craniofacial surgery. However, ongoing challenges have limited the routine use of these bone graft substitutes. Low responsiveness to BMP in humans as compared with rodents and the consequent requirement of a high dose of BMP are some of the problems.2– 4,7,11,14 Experimental data have shown that milligram quantities of rhBMP-2 are required to elicit ectopic new bone in both primates and humans (data not shown). Therefore, an improvement in the responsiveness to BMP or performance of BMP itself is an important issue for its effective and economical use in the clinical setting. We have recently demonstrated that long-term administration of a general phosphodiesterase inhibitor, Pentoxifylline (PTX), and the phosphodiesterase-4 (PDE-4)-selective inhibitor, Rolipram, could increase bone mass in normal mice by enhancing physiological bone formation.13 This was most likely achieved by increasing cyclic nucleotide (cAMP) levels in osteogenic cells. The PDEs are a large group of structurally related isoenzymes derived from at least 11 distinct genes. They can be divided into 11 groups on the basis of their substrate specificity, selective inhibition or stimulation by cofactors, selective inhibition by standard inhibitors, and gene homology.15 Based on our previous results and a recent report from another group describing PDE-4 isomer predominantly distributed in osteoblasts,1 we examined the effect of Rolipram on BMP-dependent bone formation as one approach to improve the performance of BMP in this osteoinductive protein.
Collagen sponge disks (6 mm diameter, 1 mm thickness) were impregnated with recombinant human bone morphogenetic protein-2 (rhBMP-2) (5 g/disk) and implanted onto the back muscles of mice. Ten or 20 mg/kg per day of Rolipram, a selective inhibitory agent to phosphodiesterase type 4 (PDE-4), or vehicle, was injected subcutaneously into the host mice for 3 weeks. After treatment, rhBMP-2-induced ectopic ossicles were harvested and examined by radiographic and histologic methods to determine the size, bone quality, and mineral content of the ossicles. The ossicles from a group treated with 20 mg/kg per day Rolipram were significantly larger in size and higher in bone mineral density (BMD) and bone mineral content (BMC) than the control samples. No significant differences were noted in mice treated with 10 mg/kg per day of Rolipram. Histologically, ossicles from the high-dose (20 mg/kg per day) Rolipram-treated group showed densely packed, thicker trabeculae when compared with those from the control group. These experimental results indicate that the PDE-4 inhibitor, Rolipram, may enhance the bone-inducing capacity of BMP-2 in mesenchymal cells. This in turn may result in increased responsiveness to BMP-2 and point to a potential use of PDE-4 inhibitors for the promotion of rhBMP-dependent bone repair. (Bone 30: 589 –593; 2002) © 2002 by Elsevier Science Inc. All rights reserved. Key Words: Bone morphogenetic protein (BMP); Ectopic bone formation; Rolipram; Phosphodiesterase-4 (PDE-4) inhibitor. Introduction
Materials and Methods
Fracture and the regenerative repair of bone defects associated with tumor surgery are often impaired due to inadequate fixation, extensive soft tissue damage, and the size of the bone defect and/or infection. For treatment of these types of cases, autogenous (vascularized or free graft) bone grafting has been routinely indicated despite significant disadvantages such as functional and cosmetic morbidity at the donor site and a limited source of donor bone. One of the most promising approaches for overcoming the disadvantages of autografting involves the use of bioma-
Preparation of BMP-containing Collagen Pellets rhBMP-2 was produced by the Genetics Institute (Cambridge, MA) and donated to us through Yamanouchi Phamaceutical Co. (Tokyo, Japan). The rhBMP-2 was provided in a buffer solution (5 mmol/L glutamic acid, 2.5% glycine, 0.5% sucrose, and 0.01% Tween-80) at a concentration of 1 G/L after filter sterilization. To prepare one implant sample, 5 L (5 g of rhBMP) of the rhBMP-2 solution was added to 20 L of 0.01N HCl solution and blotted onto a collagen sponge disk (6 mm diameter, 1 mm thickness) fabricated from commercially available bovine collagen sheets (Helistat, Integra Life Sciences Corp., Plainsboro, NJ), freeze-dried, and kept at ⫺20°C until
Address for correspondence and reprints: Dr. Hiroshi Horiuchi, Department of Orthodpaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, Japan. E-mail: [email protected]. shinshu-u.ac.jp © 2002 by Elsevier Science Inc. All rights reserved.
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implantation into mice. All procedures were carried out under sterile conditions. Rolipram Rolipram was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in physiological saline prior to use. Experimental Protocols Thirty male ddy mice, 4 weeks of age, were purchased from Nippon SLC Co. (Shizuoka, Japan) and housed in cages with free access to food and water for 1 week. Prior to surgery to implant the collagen sponge disks with rhBMP-2, the mice were anesthetized with diethylether. The pellets were implanted into the left dorsal muscle pouches (one pellet per animal) and divided into three groups depending on the dose of Rolipram. Zero (vehicle alone), 10 mg/kg body weight (BW)/day (low-dose Rolipram; LR), or 20 mg/kg BW/day (high-dose Rolipram; HR), of Rolipram was injected subcutaneously for 3 weeks. At the end of this period, the implants were harvested to evaluate size, bone mineral density (BMD), and bone mineral content (BMC) of the respective BMP-induced ossicle. The volume of the each ossicle was determined based on three mutually orthogonal measurements (a, b, and c) of the ossicle. These measurements were made using a caliper. The formula, V ⫽ abc/6, was used to compute ossicle volumes.7,10 All harvested tissues were radiophotographed with a soft X-ray apparatus (Sofron Co., Ltd., Tokyo, Japan). To quantitate bone quality and mass of the ossicles, BMD (milligrams per square centimeter) and BMC (milligrams) of each ossicle were measured by single-energy X-ray absorptiometry (SXA) using a bone mineral analyzer (DCS-600R, Aloka Co., Tokyo). The harvested tissues from each group were then fixed in neutralized 10% formalin, defatted in chloroform, decalcified with 10% ethylenediamine tetraacetic acid (EDTA), and embedded in paraffin wax. Sections of 5 m in thickness were cut, stained with hematoxylin-eosin, and examined under a light microscope. At 0 (control), 1, 2, and 3 weeks after implantation, blood was collected from the Rolipram-treated mice (20 mg/kg per day) and stored at ⫺20°C until biochemical measurements were performed. Serum osteocalcin was measured by immunoradiometric assay (IRMA) using a commercial kit (Immutopics, Inc., San Clemente, CA). All procedures for the animal experiments were carried out in compliance with the guidelines of the institutional animal care committee of Shinshu University.
Figure 1. Volume of harvested tissues. Volume level in the Rolipram 20 mg/kg per day group (HR) was significantly larger than in the control group (n ⫽ 10). Data expressed as mean ⫾ SD. **Significantly different from controls (p ⬍ 0.01).
significant difference vs. controls was found in the LR group (65.1 ⫾ 8.8 mm3). Radiographic Findings Areas of calcified mass on the radiograms from the HR group were significantly larger than those observed in the control group. The trabeculae in the ossicles of the HR group were more densely packed than those in the control ossicles (Figure 2). Bone Mineral Density of Ossicles Figure 3 shows the mean BMD of the ossicles from each of the groups. There was no significant difference in BMD between the LR group (15.9 ⫾ 3.6 mg/cm2) and the control group (14.5 ⫾ 2.5 mg/cm2). However, the BMD of the HR group (21.0 ⫾ 3.5 mg/cm2) was significantly higher than that of the control group.
Statistical Analysis Quantitative data were expressed as the mean ⫾ SD. Differences between experimental groups were considered statistically significant at p ⬍ 0.05 using Student’s t-test. Results Mass of rhBMP-2-induced Ossicles Figure 1 shows the mean mass of ossicles from the three groups at 3 weeks after implantation. The mean harvested bone mass of the control group was 53.1 ⫾ 7.5 mm3. The bone mass from the high-dose Rolipram (HR) group was 82.6 ⫾ 9.0 mm3 and significantly higher than that found in the control group (p ⬍ 0.01). The daily injection of a high dose of Rolipram (20 mg/kg per day) resulted in the formation of ectopic ossicles 1.5 times larger in size and with densely packed trabeculae. However, no
Figure 2. Soft X-ray photograph of the ossicle formed at 3 weeks after implantation. A typical implant from each group is shown. (A) Control group. (B) Rolipram 10 mg/kg per day group. (C) Rolipram 20 mg/kg per day group. The radio-opaque areas of the Rolipram 10 and 20 mg/kg per day groups are larger than that of the control group. Also, the radioopaque density of the harvested tissue from the Rolipram 10 and 20 mg/kg per day groups is higher than that of the control group. Scale bar ⫽ 6 mm.
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Figure 3. Effect of Rolipram treatment on bone mineral density (BMD). BMD from harvested tissues of the 20 mg/kg per day Rolipram group was significant higher than that of the control group (n ⫽ 10). Data presented as mean ⫾ SD. *Significantly different from controls (p ⬍ 0.05).
Bone Mineral Content of Ossicles Mean BMC values of the ossicles from each group are shown in Figure 4. The mean BMC of ossicles from the HR group (15.7 ⫾ 4.7 mg) was significantly greater than that of the control group (8.4 ⫾ 2.4 mg, p ⬍ 0.01). The BMC in the LR group (11.2 ⫾ 3.3 mg) revealed an upward trend but no significant difference when compared with the control group.
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Figure 5. Photomicrographs of the harvested tissue at 3 weeks after implantation. (hematoxylin-eosin stain; original magnification ⫻100). (A) Control. (B) Rolipram 10 mg/kg per day. (C) Rolipram 20 mg/kg per day. New bone formation with hematopoietic marrow and bony trabeculae is visible in the tissue. In the 20 mg Rolipram-treated group, there are visible increases in number and thickness and quantity of bony trabeculae when compared with the control group.
weeks of daily Rolipram treatment. However, serum osteocalcin increased significantly after 3 weeks of treatment (117.7 ⫾ 19.7% of controls; n ⫽ 10, p ⬍ 0.01).
Histology Discussion Ossicles from all three groups revealed normal bone histology with hematopoietic marrow and bony trabeculae. In the HR group, trabeculae were increased in number and thickness when compared with the control group (Figure 5). No significant qualitative difference was noted in the LR group. Remnants of the collagen carrier were seen in the center of all the ossicles. Osteocalcin Assay Figure 6 shows the percentage changes compared with baseline values of serum osteocalcin in the Rolipram-treated mice. The increase in serum osteocalcin was not significant after 1 and 2
Figure 4. The bone mineral content (BMC) of the harvested tissues at 3 weeks after surgery. BMC was measured by single-energy X-ray absorptiometry (SXA) using a bone mineral analyzer (DCS-600R, Aloka Co., Tokyo). The BMC of the harvested tissue from the HR group was significantly higher than in the control group (p ⬍ 0.01). However, there was no significant difference in BMC between the LR group and the control group (n ⫽ 10). Data expressed as mean ⫾ SD. **Significantly different from controls (p ⬍ 0.01).
We recently reported that Pentoxifylline (PTX), a nonselective inhibitor of PDEs, could affect BMP-2-induced new bone formation in the mouse model.9 In that study, PTX treatment resulted in increases in the BMP-induced Ca content and the size of the harvested tissues. In addition, in the study presented here, Rolipram, an inhibitor of cAMP-specific PDE-4, was shown to have the capacity to enhance BMP-2-dependent ectopic new bone formation. The daily injection of a high dose of Rolipram (20 mg/kg per day) resulted in a 1.5-fold increase in the size of ectopic ossicles and twofold greater calcium content. Both nonspecific PDE inhibitor (PTX) and PDE-4-specific inhibitor (Rolipram) have been found to enhance BMP-2-dependent ectopic new bone formation. These results indicate that PDE-4 is functionally a key enzyme in the regulation of BMP action in osteoblastic differentiation, especially in the early stage. This result indicates that the PDE-4 inhibitor could be useful for promotion of BMP-dependent bone repair. Ultimately, clin-
Figure 6. Percentage changes compared with baseline values in serum osteocalcin in Rolipram-treated mice. Serum osteocalcin increased significantly after 3 weeks of Rolipram treatment (117.7 ⫾ 19.7% of controls; n ⫽ 10, p ⬍ 0.01).
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ical trials will be necessary to confirm the efficacy of this approach in humans. In an initial phase of ectopic new bone formation in the BMP-retaining collagen pellets, the pellet is resorbed and replaced by cartilage shell at the periphery of the pellet. This is then replaced by new bone through endochondral ossification. The shell determines the final size of the BMP-induced ossicle. In this study, it was determined radiographically that the calcified rings in tissues harvested from mice treated with 20 mg/kg per day of Rolipram for 1 and 2 weeks were larger than those observed in the control group (data not shown). Because the chondro-osseous differentiation of the young mesenchymal cells is initiated when these cells come into contact with BMP at the periphery of the BMP-retaining disk, this increase in bone mass may indicate an increased sensitivity and earlier initiation of the response to BMP-2. Therefore, Rolipram appears to have a stimulatory effect on the early stage of bone formation during BMP-induced osteogenesis. However, histological findings showed that new bone formations were induced during each stage of endochondral bone formation in both the control and Rolipram-treated groups (data not shown). In addition, Rolipram seemed to enhance osteoblastic function as shown by the dense distribution of thick trabeculae in the Rolipram-treated ossicles. This effect may be driven through enhancement of osteoblastic function and not by suppression of bone resorption. Earlier histomorphometric observations of bone from Rolipram-treated animals showed increased bone formation.13 We have investigated the stimulatory effects of the PDE-4 inhibitor, Rolipram, on osteoblasts lineages in vitro. This in vitro study has demonstrated that Rolipram could stimulate BMPinduced chondro-osteoblast differentiation.24 It was also shown that a selective PDE-4 inhibitor enhanced BMP-4-induced alkaline phosphatase (ALP) expression in a dose-dependent manner in osteoblastic cells.24 Finally, northern blot analysis showed that Rolipram enhanced levels of expression of the mRNAs of ALP and osteopontin in osteoblastic cells.24 These results support the findings reported herein that the PDE-4 inhibitor, Rolipram, enhances BMP-induced bone formation in vivo. The general pharmacological effects of PDE inhibitors are brought about through elevated level of intracellular cyclic nucleotides (cyclic AMP or cyclic GMP), suppressed degradation of these cyclic nucleotides, and the resulting activation of protein kinase A (PKA).1 However, little is currently known about the roles of PDE or PDE inhibitors in bone metabolism, specifically in the context of BMP-responding mesenchymal cells or osteoblasts. PDEs constitute a family of enzymes encoded by at least 20 distinct genes that can be classified into 11 groups (PDE-1 through PDE-11), depending on their enzymological characteristics.5 Further complexity can be noted because several splice variants have been identified for specific PDEs, some of which are expressed in a tissue-specific manner. The details regarding PDE isomer expression in mesenchymal or osteoblastic cells are not clear. A recent report indicated predominant distribution of cAMP-specific PDE-4 activity (about 85%) in osteoblastic cells in an vitro system.1 Previous in vivo studies conducted in our laboratory have also indicated an anabolic effect of Rolipram on normal bone formation in mice.13 The present study has also identified a significant increase in serum osteocalcin levels after 3 weeks of Rolipram treatment. Rolipram was used in this study to improve the performance of rhBMP-2. The results also indicated the apparent involvement of PDE-4 in the osteogenetic process. Splicing variants of PDE-4, which are specific to mesenchymal cells or osteoblasts, if they exist, current await identification. If a PDE-4 isomer and its
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selective inhibitory agent are identified, the agent will be utilized more effectively to promote BMP-dependent bone repair. This approach could also be used in the treatment of osteoporosis by increasing bone mass through enhanced systemic bone formation without adverse effects to other organs. With regard to the mechanism of action of Rolipram or activated PKA by elevated intracellular cAMP level in cells, two possible hypothesis must be considered: (1) suppressed production of cytokines antagonistic to BMP, such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1 (IL-1),6,12,16 –23,25 and consequent enhancement of BMP action; and (2) acceleration of the intracellular BMPsignaling pathway by crosstalk with the PKA-mediated signaling pathway. More precise in vitro studies are needed to test these hypotheses. Further work is underway in our laboratory to achieve this goal.
Acknowledgment: This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (No. 12671403), Japan, and grants from the Japan Rheumatism Foundation, the Hip Joint Foundation of Japan, and the Japan Orthopaedics and Traumatology Foundation, Inc. (No. 0120).
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Date Received: April 2, 2001 Date Revised: June 15, 2001 Date Accepted: December 19, 2001