Comments for:

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Response to Baylink and colleagues

Submitter: Robert L. Jilka, Ph.D. | rljilka@life.uams.edu

University of Arkansas for Medical Sciences

Published January 18, 1999

We thank Dr. Baylink and colleagues for their interest in our work. We hope that the following will clarify the issues they have raised, and will satisfy their concerns.
The data shown in Figures 3, 6 and 8 were obtained using cells cultured from each animal separately. Inadvertently, this information was omitted from the figure legends. The variation in the number of CFU-F and CFU-OB in different experiments can be easily explained by the use of different serum lots at different stages of this work which spanned a period of over 2 years. The data of Figures 3 and 4 of course cannot prove that the increase in osteoblast progenitors initiates the increase in bone formation seen at 7 days after ovariectomy. However, the lifespan of osteoblasts is approximately 10-14 days in the mouse (1). Therefore, the sustained increase in bone formation following ovariectomy (for at least 8 weeks) can only be maintained by an increase in de novo osteoblast development, as reflected by our finding of increased CFU-OB. Be that as it may, an increase in the replication of osteoblastic cells that are beyond the colony forming stage, such as reported by Modrowski et al. in ref 31, may be responsible for the early increase in bone formation.
Circulating osteocalcin reflects bone formation throughout the skeleton, whereas the CFU-OB measurements of our studies reflect osteoblastogenesis in the femur. Therefore, it is not surprising that osteocalcin did not exactly parallel CFU-OB in alendronate-treated sham- operated animals, especially since a portion of circulating osteocalcin must be originating from osteoblasts at bone formation sites undergoing modeling – a process that is ongoing in 10-12 week old mice and of course does not require previous bone resorption. In full agreement with our findings, an increase in osteoclast formation in alendronate-treated mice has also been documented by van Beek et al in reference 38 of the paper.
All quibbling aside, there is overwhelming evidence that not only is osteoblastogenesis upregulated following ovariectomy, but osteoblastogenesis is a prerequisite for the upregulation of osteoclastogenesis, as the two processes are intimately linked at the early stage of differentiation, not at the functional level as previously thought (2-9). The recent demonstration of the requirement of an osteoblast-specific transcription factor (Cbfa1) for the expression of the RANKL gene, a sine qua non for osteoclastogenesis, provides the ultimate proof for this concept by elucidating the molecular basis of the linkage between the two processes (10). It is very likely, however, that this tonic baseline control of osteoblastogenesis and osteoclastogenesis can be influenced by growth factors released during resorption.
Robert L. Jilka, Ph.D.
Robert S. Weinstein, M.D.
Stavros C. Manolagas, M.D., Ph.D.
Div. of Endocrinology and Metabolism
Center for Osteoporosis and Metabolic Bone Diseases
Veterans Affairs Medical Center
University of Arkansas for Medical Sciences
Little Rock, AR

References
1. Jilka, R.L., R.S. Weinstein, T. Bellido, A.M. Parfitt, and S.C. Manolagas. 1998. Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J.Bone Miner.Res. 13:793-802.
2. Weinstein, R.S., R.L. Jilka, A.M. Parfitt, and S.C. Manolagas. 1997. The effects of androgen deficiency on murine bone remodeling and bone mineral density are mediated via cells of the osteoblastic lineage. Endocrinology 138:4013-4021.
3. Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.-H. Gao, M. Inada, M. Sato, T. Okamoto, Y. Kitamura, S. Yoshiki, and T. Kishimoto. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755-764.
4. Gao, Y.H., T. Shinki, T. Yuasa, H. Kataoka-Enomoto, T. Komori, T. Suda, and A. Yamaguchi. 1998. Potential role of Cbfa1, an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis: Regulation of mRNA expression of osteoclast differentiation factor (ODF). Biochem. Biophys. Res. Comm. 252:697-702.
5. Lacey, D.L., E. Timms, H.L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, H. Hsu, J. Sullivan, N. Hawkins, E. Davy, C. Capparelli, A. Eli, Y.X. Qian, S. Kaufman, I. Sarosi, V. Shalhoub, G. Senaldi, J. Guo, J. Delaney, and W.J. Boyle. 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165-176.
6. Yasuda, H., N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinoshaki, S. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, and T. Suda. 1998. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc.Nat.Acad.Sci. USA 95:3597-3602.
7. Boyle, W.J., Y. Kung, D.L. Lacey, I. Sarosi, C. Dunstan, E. Timms, H.-L. Tan, G. Elliott, M.J. Kelley, A. Colombero, R. Elliott, S. Scully, C. Capparelli, S. Morony, and J. Penninger. 1998. Osteoprotegerin ligand (OPGL) is required for murine osteoclastogenesis. Bone 23:S189(Abstr.)
8. Corral, D.A., M. Amling, M. Priemel, E. Loyer, S. Fuchs, P. Ducy, R. Baron, and G. Karsenty. 1998. Dissociation between bone resorption and bone formation in osteopenic transgenic mice. Proc.Natl.Acad.Sci.USA 95:13835-13840.
9. Abe, E., M. Yamamoto, Y. Taguchi, B. Lecka-Czernik, A.N. Economides, N. Stahl, R.L. Jilka, and S.C. Manolagas. 1998. Requirement of BMPs 2/4 for postnatal osteoblast as well as osteoclast formation: antagonism by noggin. Bone 23:S242(Abstr.)
10. O'Brien, C.A., N. Farrar, and S.C. Manolagas. 1998. Identification of an OSF-2 binding site in the murine RANKL/OPGL gene promoter: a potential link between osteoblastogenesis and osteoclastogenesis. Bone 23:S149(Abstr.)

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Concerns about the conclusions of Jilka et al.

Submitter: David J. Baylink, M.D. | baylinkd@llvamc.va.gov

Jerry L. Pettis VA Medical Center

Published January 18, 1999

The paper by Dr. Jilka, et al. (1) in the May issue of the Journal of Clinical Investigation addressed a very important subject: The mechanism for the coupling of bone formation to bone resorption. These investigators found an increase in colony-forming unit osteoblast (CFU-OB) in marrow cell cultures prepared from ovariectomized mice. They interpreted this as evidence of an osteogenic response to either bone resorption or estrogen deficiency. Because their studies also showed that alendronate decreased bone resorption in the ovariectomized mice, but did not abolish the increase in CFU-OB, the authors also concluded that the osteogenic response (i.e., the increase in CFU-OB) was not due to the release of growth factors from bone during bone resorption (2, 3), but rather, due to estrogen deficiency. While we are intrigued by this provocative hypothesis, we are also concerned with a number of apparent internal inconsistencies in some of the data presented - inconsistencies which could affect the conclusions.
1) The increase in osteoclastogenesis was seen within 7 days of ovariectomy and this was attributed to estrogen deficiency. The increase in serum osteocalcin was also seen within 7-14 days, and this was interpreted as evidence of increased osteoblast activity (i.e., increased bone formation). Together, these observations suggested parallel actions of estrogen deficiency on bone formation and resorption. Although the increase in the number of osteoblast progenitor cells in the marrow (CFU- OB) was not seen until 28 days after ovariectomy, the authors interpret their data as indicating that the increase in serum osteocalcin and the increase in CFU-OB had similar time courses. A consideration of the data summarized in figures 4B and 5 suggests that the increase in serum osteocalcin is not correlated with the increase in CFU-OB. Furthermore, it is difficult to see how the increase in osteocalcin could be interpreted as evidence of an “impact on osteoblast biology” of the changes in CFU-OB, when it preceded them by two weeks.
2) If, as the authors suggest, the increase in osteocalcin was an indication of the functional relevance of the increase in CFU-OB, we would expect that the significant difference in CFU-OB between sham and ovariectomized mice in the alendronate treated groups (Figure 8) would be mimicked by a similar difference in serum osteocalcin. This was, however, not the case.
3) The in vitro studies of osteoclastogenesis revealed significant differences in the number of osteoclasts formed from marrow cells derived from sham and ovariectomized mice, even after alendronate treatment. If this difference were biologically significant, it should also have been predictive of a parallel difference in bone resorption; however, the amount of deoxypyridinoline in urine (an index of systemic bone resorption) did not change in parallel (Figure 8).
These inconsistencies are troubling - the increase in osteocalcin preceded the increase in CFU-OB and, therefore, could not have been its consequence, and the authors did not observe parallel changes in the biochemical markers of bone turnover and the numbers of osteoclasts and CFU-OB in the alendronate-treated mice. Since further studies will be required to resolve these issues, the authors may wish to consider the following aspects of study design. First, the in vitro studies were limited to assessments of bone marrow cells derived from a single bone – the femur – which may not be representative of the entire skeleton, whereas the biochemical markers of bone turnover reflect changes in the entire skeleton. The use of pooled samples precludes an assessment of variation between mice within a treatment group and this is potentially confounding. Perhaps, this aspect of study design led to some of the large variations and inconsistencies in the results. For example, the authors advise us that the number of osteoblast progenitor cells per femur ranged from 400 to 2,500 for colony-forming unit fibroblast (CFU-F) and 100 to 800 for CFU-OB, in the sham-operated group. These are large variations. Furthermore, the results of replicate assessments of CFU-F (in Figure 4A) indicate a variation on the order of 100%. Third, an effect of ovariectomy on CFU-F was observed at 14 days in the first experiment, but not in the second. It is difficult to interpret the significance of observed differences in the face of the above mentioned variations and internal inconsistencies.
Regarding the conclusion that estrogen deficiency results in an increase in bone formation that is independent of prior bone resorption (and, therefore, unaffected by the release of growth factors from bone), it is interesting to note that the biologically relevant data are also consistent with the opposite conclusion. Despite the delayed increase in CFU-OB after ovariectomy, the effects of alendronate treatment on the biochemical markers (decreased deoxypyridinoline and decreased osteocalcin) are entirely consistent with previous findings from other studies – a decrease in bone resorption, followed by a coupled decrease in bone formation (4, 5). Moreover, since the changes in CFU-OB are not concordant with the changes in osteocalcin, and have not been shown to result in an eventual increase in the rate of bone formation, they may not be relevant to the biological phenomenon of coupled bone formation, in response to bone resorption.
In conclusion, the authors have developed an interesting model to evaluate bone turnover parameters. This model has the potential to disclose important insights into the mechanism of coupling at the cell and molecular levels. This system also has the potential for misinterpretation, because it involves a comparison of in vivo observations with in vitro data from long-term marrow cell cultures. We would, therefore, encourage the investigators to regard their conclusions as provisional, and to pursue their interesting findings with additional experiments.
David J. Baylink, M.D.
John Farley, Ph.D.
K-H William Lau, Ph.D.
Subburaman Mohan, Ph.D.
Loma Linda University
Jerry L. Pettis VA Medical Center
11201 Benton Street
Loma Linda, CA 92357
(909) 422-3101
FAX (909) 796-1680
E-mail: baylinkd@llvamc.va.gov
References
1. Jilka RL, Takahashi K, Munshi M, Williams DC, Roberson PK and Manolagas SC. 1998. Loss of Estrogen Upregulates Osteoblastogenesis in the Murine Bone Marrow: Evidence for Autonomy from Factors Released during Bone Resorption. J. Clin. Invest. 101:1942-1949.
2. Farley JR, Tarbaux N, Murphy LA, Masuda T and Baylink DJ. 1987. In Vitro Evidence That Bone Formation May Be Coupled to Resorption by Release of Mitogen(s) From Resorbing Bone Metabolism36:314-321.
3. Pfeilschifter J and Mundy GR. 1987. Modulation of type transforming growth factor activity in bone cultures by osteotropic hormones. Proc. Natl. Acad. Sci. USA84:2024-2028.
4. Baylink DJ, Jennings JC and Mohan S. 1998. Calcium and bone homeostasis and changes with aging. InPrinciples of Geriatric Medicine and Gerontology. W.R. Hazzard, J.P. Blass, W.H. Ettinger, J.B. Halter, J.G. Ouslander, editors. McGraw Hill, New York. NY 1041-1056.
5. Garnero P, Shih WJ, Gineyts E, Karpf DB and Delmas PD. 1994. Comparison of New Biochemical Markers of Bone Turnover in Late Postmenopausal Osteoporotic Women in Response to Alendronate Treatment. J. Clin. Endocrinol. Metab.79:1693-