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Required and nonessential functions of nuclear factor-kappa B in bone cells
Bone Vol. 25, No. 1 July 1999:137–139
Required and Nonessential Functions of Nuclear Factor-Kappa B in Bone Cells B. F. BOYCE,1 L. XING,1 G. FRANZOSO,2 and U. SIEBENLIST1 1
Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA Laboratory of Immunoregulation, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA
2
Introduction
Nuclear factor-kappa B (NF-kB) is a set of five polypeptide transcription factors, called p50, p52, p65 (also called Rel A), Rel B, and c-Rel, which regulate the expression of a variety of genes involved in immune and inflammatory responses. They were originally named because they were considered essential regulators of B cell kappa light chain expression. More recent studies indicate that NF-kB proteins are involved in the regulation of a variety of other cell functions, including cell proliferation, responses to stress, and apoptosis. NF-kB heterodimers reside in the cytoplasm of cells bound to inhibitory proteins, the two commonest of which are IkBa and IkBb, which prevent NF-kB from entering the nucleus. When cells are stimulated, IkB is phosphorylated by specific IkB kinases and subsequently is ubiquitinated and degraded in proteosomes. This allows NF-kB to translocate to the nucleus to regulate the expression of a growing list of genes, including the proinflammatory cytokines, interleukin-1 (IL-1), IL-6, and tumor necrosis factor. IL-1 and tumor necrosis factor in turn also regulate the expression of NF-kB. Thus, once activated, NF-kB may be involved in upregulatory loops, which can amplify the effects of the initiating stimulus. Because these proinflammatory cytokines have been implicated in the pathogenesis of estrogen deficiency and inflammation-related bone loss, it is likely that NF-kB has a significant role in the increased generation and function of osteoclasts in these circumstances. However, an unexpected and essential role of NF-kB in the formation of osteoclasts during development was discovered recently after the generation of knockout mice, which lack the expression of the p50 and p52 subunits. This paper will describe recent studies that reveal an essential role for NF-kB signaling in the generation of osteoclasts and that suggest that NF-kB may also play a key central role in the activation and survival of osteoclasts in conditions in which osteoclastogenesis is upregulated. (Bone 25: 137–139; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Knockout of the five individual nuclear factor-kappa B (NF-kB) proteins resulted in anticipated defects in immune cell function (p50, p52, Rel B, and c-Rel knockout mice; reviewed in ref. 19) or in embryonic death (p652/2 mice had massive liver cell apoptosis at embryonic day 11).4 Because p50 and p65 are the predominant heterodimers formed in most situations when NF-kB is activated, it was anticipated that generation of p502/2, p522/2 double-knockout mice (dKO) would eliminate most NF-kB signaling in the animals. Generation of p502/2, p522/2 dKO mice resulted in the live birth of mice that appeared indistinguishable from their wild-type (WT) littermates at birth. However, the incisors of the dKO mice did not erupt and the animals failed to thrive after weaning,7,12 most of them failing to survive beyond 4 –5 weeks of age, when their average body weight (10 –12 g) is about 30% of that of WT littermates. Radiologic and histologic examination of bones from the dKO animals revealed severe osteopetrosis.7,12 Osteopetrosis is a disorder of bone modeling that can occur due to failure of osteoclast formation or function.23 Examination of tartrate-resistant acid phosphatase (TRAP)-stained sections revealed almost complete absence of TRAP1 cells in bone sections from the dKO mice. Failure of osteoclast formation can result from defective production of osteoclastogenic signals from stromal cells or from defects within the osteoclast lineage, the latter of which typically can be rescued by transfer of hematopoietic precursors to osteopetrotic mice.23 Transplantation of hematopoietic precursors from embryonic WT liver or bone marrow cells to irradiated neonatal NF-kB dKO mice rescued the defect in osteoclastogenesis and resulted in tooth eruption and prevention of osteopetrosis.7,12 Normal numbers of TRAP1 osteoclasts were observed in bone sections from the transplanted mutant mice that gained body weight and thrived normally until death at 3 months after birth. To confirm that the defect in osteoclastogenesis in the NF-kB dKO mice is in the osteoclast lineage, coculture experiments using osteoblasts and spleen cells were carried out using standard techniques.18 Coculture of NF-kB dKO calvarial osteoblasts supported the formation of osteoclasts from precursors in spleen cells from WT mice. In contrast, in cocultures of WT calvarial osteoblasts and spleen cells from NF-kB dKO mice, no TRAP1 cells, either mononuclear or multinucleated were formed.7,12 These in vitvo and in vivo observations indicate that expression of the p50 and p52 subunits of NF-kB is required for osteoclast formation during development and that the defect resides within cells in the osteoclast lineage.
Key Words: NF-kB; Osteopetrosis; Osteoclasts; Lymphocytes; Cytokines.
Address for correspondence and reprints: Brendan F. Boyce, M.D., Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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Previous studies of naturally occurring or genetically engineered forms of osteopetrosis have indicated that expression of the transcription factor, PU.1, is required for commitment of precursors to the myelomonocyte lineage29 and that expression of c-fos10,30 and macrophage colony-stimulating factor (MCSF)28,34 is required for commitment of monocyte precursors to the osteoclast lineage. c-fos2/2 and op/op mice form no or very few osteoclasts, but they do produce macrophages, which are present in increased numbers in the marrow cavities of c-fos mutants. Examination of spleen and liver sections revealed that NF-kB dKO mice have normal or increased numbers of macrophages stained using the antimacrophage antibody F4/80.7 Macrophage numbers are also increased in bone marrow,12 indicating that NF-kB functions at a point in the osteoclast lineage very close to that at which c-fos expression is necessary. However, expression of c-fos in the NF-kB dKO mice is normal.12 Thus, both of these transcription factors have critical, nonoverlapping functions in the regulation of genes that commit precursors to the osteoclast lineage at a stage before TRAP is expressed and beyond the point at which monocyte differentiation to macrophages occurs. Since NF-kB regulates the expression of the osteoclastogenic cytokines, interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage (GM)-CSF,5,25 it is possible that osteoclasts fail to form in the NF-kB dKO mice because these cytokines do not get expressed. To determine if expression of these cytokines was normal in NF-kB dKO mice, peritoneal macrophages were elicited from the dKO mice, stimulated with lipopoly saccharide (LPS), and mRNA was extracted. Basal levels of expression of IL-6, IL-1a, and b, and TNF were mostly normal, and expression increased in response to LPS in a manner similar to that seen in WT littermates.7,12 However, expression of IL-6 protein was reduced in dKO macrophages under basal and stimulated conditions.12 Although mRNA expression of these cytokines appears to be normal in peritoneal macrophages from the NF-kB dKO mice, it is possible that production of these proteins by stromal cells or by cells in the osteoclast lineage is defective. To determine if the defect in osteoclast formation could be rescued in vitro by addition of these cytokines, we treated cocultures of osteoblasts and spleen cells from NF-kB dKO mice with IL-1, IL-6, IL-6, and soluble IL-6 receptor (sIL-6R) or GM-CSF alone and in combination. No TRAP1 mononuclear or multinucleated cells were detected in cultures treated with IL-6, IL-1, TNF, or GM-CSF. However, small numbers of TRAP1 multinucleated and mononuclear cells were detected in cultures treated with IL-6 1 sIL-6R.32 Occasional TRAP1 multinucleated cells are also observed in bone sections from NF-kB dKO mice,7 but we do not yet know if these cells or the TRAP1 cells formed in vitro after treatment with IL-6 1 sIL-6R have the capacity to resorb bone. However, if these cells and the multinucleated cells formed when WT osteoblasts and spleen cells from NF-kB dKO mice are treated with IL-6 1 sIL-6R can resorb bone, our findings suggest that some osteoclasts can form in the absence of NF-kB p50 and p52 signaling. We do not know yet if the in vivo mechanism for occasional osteoclast formation is upregulated by IL-6 1 sIL-6 R. It will be important to determine if formation of these TRAP1 cells is mediated by other members of the NF-kB family or if it occurs by an NF-kB-independent mechanism. Recent studies have indicated that osteoclast formation in vitro requires expression of RANK (receptor activator of NF-kB) ligand (RANKL),1 also known as ODF,33 TRANCE,31 and osteoprotegerin (OPG) ligand,17 along with M-CSF by osteoblasts/stromal cells. RANKL binds to its receptor, RANK, on the surface of TRAP1 osteoclast precursors.17 Thus, the failure of osteoclast formation in the NF-kB dKO mice could be due to the
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absence of NF-kB signaling after RANKL/RANK interaction, or because NF-kB dKO mice do not express RANK or c-fms, the tyrosine kinase receptor for M-CSF. c-fms and RANK are expressed in NF-kB dKO mouse spleen cells at levels similar to those in WT mice.32 However, it remains to be shown that the RANK-expressing cells in spleens of NF-kB dKO mice are osteoclast precursors, rather than cells in other lineages, before it can be concluded definitively that the defect is downstream of RANKL/RANK interaction. RANKL has also been shown to stimulate the resorptive activity of isolated osteoclasts in vitro,8,17 suggesting that if RANKL/RANK signaling in these circumstances is mediated through NF-kB, NF-kB may have critical regulatory roles at multiple stages of osteoclast function and activity. NF-kB could also play a significant role in the regulation of the increased osteoclastogenesis associated with estrogen deficiency and inflammatory conditions affecting bone in which expression of the osteoclastogenic cytokines, IL-1, IL-6, and TNF, are increased.11,14,21 NF-kB has been implicated in postmenopausal bone loss because it regulates IL-6 expression through its interaction with the estrogen receptor (ER).9,26 After binding of estrogen to its receptor in osteoblasts, the ER interacts with NF-kB heterodimers after they have been released from the inhibitory binding protein, IkB.9,26 This interaction with the ER prevents NF-kB from binding to response elements in the IL-6 promoter and activating gene expression. Thus, osteoclastogenesis may be kept in check in states of estrogen repletion as a result of this inhibitory effect on IL-6 expression. After the menopause, the inhibitory effect of estrogen and ER interaction with NF-kB is withdrawn, and increased osteoclastogenesis may ensue as a consequence of enhanced IL-6, IL-1, and TNF production by bone marrow macrophages through regulatory loops2,3 in which NF-kB activation leads to enhanced production of these cytokines and more NF-kB activation with subsequent increased production of RANKL and M-CSF by stromal cells. Production of IL-1, IL-6, and TNF by synovial cells and macrophages is increased in patients with rheumatoid arthritis (reviewed in refs. 13 and 27) and may account for the increased osteoclastogenesis and bone loss seen around affected joints in patients with this disorder. NF-kB may play in a role in this increased osteoclastogenesis by activating the expression of these cytokines locally in tissues of affected joints. At present, it is not known which NF-kB proteins might be involved in regulation of the increased osteoclastogenesis in these disorders. NF-kB may also regulate osteoclast survival. Recent studies have indicated that treatment of isolated osteoclasts with inhibitors of NF-kB20 or with antisense oligonucleotides15 to the p65 or p50 subunits of NF-kB induces apoptosis. We have treated osteoclasts generated from bone marrow cultures with the NF-kB inhibitors, TPCK, PDTC, and MG-132, and have found that they promote apoptosis of these cells at doses that have been shown to prevent translocation of NF-kB from the cytoplasm to the nucleus. However, apoptosis of stromal cells was also observed in these cultures at these concentrations and also at significantly lower concentrations. Our previous studies indicate that osteoblast/stromal cell function in the NF-kB dKO mice is normal (they are able to support osteoclast formation from WT spleen cells); bone formation is unimpaired in NF-kB dKO mice and there is no evidence of increased osteoblast apoptosis in bone from the dKO mice (B. F. Boyce, unpublished observation). Thus, the increased osteoclast apoptosis induced by these agents may be nonspecific and mediated through a mechanism that might not involve NF-kB signaling. Finally, NF-kB also appears to have a role in limb development. Limb formation occurs as a result of interaction between mesenchynal and overlying epithelial cells in the apical epider-
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mal ridge in the primitive limb bud (reviewed in ref. 22). Inhibition of NF-kB c-Rel expression in mesenchymal cells results in reduction of limb size and failed development of the bones of the hands and feet, because NF-kB-activated FGF10 production does not occur.6,16 This, however, is a nonessential function of c-Rel because limb development in c-Rel knockout mice is normal. Since the original observation that NF-kB signaling was involved in kappa light chain production,24 many functions have been identified for NF-kB in numerous cell types. So far, essential functions for NF-kB p50 and p52 proteins have been identified for the formation osteoclasts and mature B cells.7 It is possible that these or other members of the NF-kB family of proteins have important, though nonessential, regulatory roles in other bone cell types, which may be revealed by future studies of osteoblasts and chondrocytes from these or other NF-kB knockout animals. References 1. Anderson, D. M., Maraskovsky, E., Billingsley, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179; 1997. 2. Baldwin, A. S., Jr. The NF-kB and IkB proteins: new discoveries and insights. Annu Rev Immunol 14:649 – 83; 1996. 3. Barnes, P. J. and Karin, M. Nuclear factor-kB-a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066 –1071; 1997. 4. Beg, A., Sha, W., Bronson, R., Ghosh, S., and Baltimore, D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kB. Nature 376:167–170; 1995. 5. Beg, A. A. and Baltimore, D. An essential role for NF-kB in preventing TNF-a-induced cell death. Science 274:782–784; 1996. 6. Bushdid, P. B., Brantley, D. M., Yull, F. E., et al. Inhibition of NF-kB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis. Nature 392:615– 618; 1998. 7. Franzoso, G., Carlson, L., Xing, L., et al. Requirement for NF-kB in osteoclast and B cell development. Genes Dev 11:3482–3496; 1997. 8. Fuller, K., Wong, B., Fox, S., Choi, Y., and Chambers, T. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001; 1998. 9. Galien, R. and Garcia, T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Nucleic Acids Res 25: 2424 –2429; 1997. 10. Grigoriadis, A. E., Wang, Z.-Q., Cecchini, M. G., et al. c-Fos: A key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266:443– 448; 1994. 11. Horowitz, M. C. Cytokines and estrogen in bone; anti-osteoporotic effects. Science 260:626 – 627; 1993. 12. Iotsova, V., Caamano, J., Loy, J., Lewin, A., and Bravo, R. Osteopetrosis in mice lacking NF-kB1 and NF-kB2. Nature Med 3:1285–1289; 1997. 13. Isomaki, P., and Punnonen, J. Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann Med 29:499 –507; 1997. 14. Jilka, R. L. Cytokine, bone remodeling, and estrogen deficiency. J Bone Min Res 23:75– 81; 1998.
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15. Jimi, E., Nakamura, I., Ikebe, T., Akiyama, S., Takahashi, N., and Suda, T. Activation of NF-kB is involved in the survival of osteoclasts promoted by Interleukin-1. J Biol Chem 273:8799 – 8805; 1998. 16. Kanegae, Y., Tavares, A. T., Izpisua-Belmonte, J. C., and Verma, I. M. Role of rel/NFkB transcription factors during the outgrowth of the vertebrate limb. Nature 392:611– 614; 1998. 17. Lacey, D. L., Timms, E., Tan, H. L., et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176; 1998. 18. Lowe, C., Yoneda, T., Boyce, B. F., Chen, H., Mundy, G. R., and Soriano, P. Osteopetrosis in src-deficient mice is due to an autonomous defect of osteoclasts. Proc Natl Acad Sci USA 90:4485– 4489; 1993. 19. May, M. J. and Ghosh, S. Signal transduction through NF-kB. Immunology Today 19:80 – 88; 1998. 20. Ozaki, K., Takeda, H., Iwahashi, H., Kitano, S., and Hanazawa, S. NF-kB inhibitors stimulate apoptosis of rabbit mature osteoclasts and inhibit bone resorption by these cells. Fed Euro Biochem Soc 410:297–300; 1997. 21. Pacifici, R. Cytokines, estrogen, and postmenopausal osteoporosis—the second decade. Endocrinology 139:2659 –2661; 1998. 22. Panganiban, G., Irvine, S., Lowe, C., et al. The origin and evolution of animal appendages. Proc Natl Acad Sci USA 94:5162–5166; 1997. 23. Popoff, S. N. and Schneider, G. B. Animal models of osteopetrosis: the impact of recent molecular developments on novel strategies for therapeutic intervention. Mol Med Today 2:349 –358; 1996. 24. Sen, R. and Baltimore, D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705–716; 1986. 25. Siebelist, U., Franzoso, G. and Brown, K. Structure, regulation and function of NF-kB. Annu Rev Cell Bio 10:405– 455; 1994. 26. Stein, B. and Yang, M. X. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Mol Cell Biol 15:4971– 4979; 1995. 27. Szekanecz, Z., Koch, A., Kunkel, S., and Strieter, R. Cytokines in rheumatoid arthritis. Potential targets for pharmacological intervention. Drugs and Aging 12:377–390; 1998. 28. Takahashi, N., Udagawa, N., Akatsu, T., Tanaka, H., Isogai, Y., and Suda, T. Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128:1792– 1796; 1991. 29. Tondravi, M. M., McKercher, S. R., Anderson, K., et al. Osteopetrosis in mice lacking haematopoietic transcription factor PU.1. Nature 386:81– 84; 1997. 30. Wang, Z.-Q., OVitt, C., Grigoriadis, A. E., Mohle-Steinlein, U., Ruther, U., and Wagner, E. F. Bone and haematopoietic defects in mice lacking c-Fos. Nature 360:741–745; 1992. 31. Wong, B. R., Rho, J., Arron, J., et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 272:25190 –25194; 1997. 32. Xing, L. P., Carlson, L., Siebenlist, U., and Boyce, B. F. Mechanisms by which NF-kB regulates osteoclast numbers. Bone 23(Suppl.):190; 1998. 33. Yasuda, H., Shima, N., Nakagawa, N., et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602; 1998. 34. Yoshida, H., Hayashi, S., Kunisada, T., et al. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442– 444; 1990.
Required and Nonessential Functions of Nuclear Factor-Kappa B in Bone Cells B. F. BOYCE,1 L. XING,1 G. FRANZOSO,2 and U. SIEBENLIST1 1
Department of Pathology, University of Texas Health Science Center, San Antonio, TX, USA Laboratory of Immunoregulation, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA
2
Introduction
Nuclear factor-kappa B (NF-kB) is a set of five polypeptide transcription factors, called p50, p52, p65 (also called Rel A), Rel B, and c-Rel, which regulate the expression of a variety of genes involved in immune and inflammatory responses. They were originally named because they were considered essential regulators of B cell kappa light chain expression. More recent studies indicate that NF-kB proteins are involved in the regulation of a variety of other cell functions, including cell proliferation, responses to stress, and apoptosis. NF-kB heterodimers reside in the cytoplasm of cells bound to inhibitory proteins, the two commonest of which are IkBa and IkBb, which prevent NF-kB from entering the nucleus. When cells are stimulated, IkB is phosphorylated by specific IkB kinases and subsequently is ubiquitinated and degraded in proteosomes. This allows NF-kB to translocate to the nucleus to regulate the expression of a growing list of genes, including the proinflammatory cytokines, interleukin-1 (IL-1), IL-6, and tumor necrosis factor. IL-1 and tumor necrosis factor in turn also regulate the expression of NF-kB. Thus, once activated, NF-kB may be involved in upregulatory loops, which can amplify the effects of the initiating stimulus. Because these proinflammatory cytokines have been implicated in the pathogenesis of estrogen deficiency and inflammation-related bone loss, it is likely that NF-kB has a significant role in the increased generation and function of osteoclasts in these circumstances. However, an unexpected and essential role of NF-kB in the formation of osteoclasts during development was discovered recently after the generation of knockout mice, which lack the expression of the p50 and p52 subunits. This paper will describe recent studies that reveal an essential role for NF-kB signaling in the generation of osteoclasts and that suggest that NF-kB may also play a key central role in the activation and survival of osteoclasts in conditions in which osteoclastogenesis is upregulated. (Bone 25: 137–139; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Knockout of the five individual nuclear factor-kappa B (NF-kB) proteins resulted in anticipated defects in immune cell function (p50, p52, Rel B, and c-Rel knockout mice; reviewed in ref. 19) or in embryonic death (p652/2 mice had massive liver cell apoptosis at embryonic day 11).4 Because p50 and p65 are the predominant heterodimers formed in most situations when NF-kB is activated, it was anticipated that generation of p502/2, p522/2 double-knockout mice (dKO) would eliminate most NF-kB signaling in the animals. Generation of p502/2, p522/2 dKO mice resulted in the live birth of mice that appeared indistinguishable from their wild-type (WT) littermates at birth. However, the incisors of the dKO mice did not erupt and the animals failed to thrive after weaning,7,12 most of them failing to survive beyond 4 –5 weeks of age, when their average body weight (10 –12 g) is about 30% of that of WT littermates. Radiologic and histologic examination of bones from the dKO animals revealed severe osteopetrosis.7,12 Osteopetrosis is a disorder of bone modeling that can occur due to failure of osteoclast formation or function.23 Examination of tartrate-resistant acid phosphatase (TRAP)-stained sections revealed almost complete absence of TRAP1 cells in bone sections from the dKO mice. Failure of osteoclast formation can result from defective production of osteoclastogenic signals from stromal cells or from defects within the osteoclast lineage, the latter of which typically can be rescued by transfer of hematopoietic precursors to osteopetrotic mice.23 Transplantation of hematopoietic precursors from embryonic WT liver or bone marrow cells to irradiated neonatal NF-kB dKO mice rescued the defect in osteoclastogenesis and resulted in tooth eruption and prevention of osteopetrosis.7,12 Normal numbers of TRAP1 osteoclasts were observed in bone sections from the transplanted mutant mice that gained body weight and thrived normally until death at 3 months after birth. To confirm that the defect in osteoclastogenesis in the NF-kB dKO mice is in the osteoclast lineage, coculture experiments using osteoblasts and spleen cells were carried out using standard techniques.18 Coculture of NF-kB dKO calvarial osteoblasts supported the formation of osteoclasts from precursors in spleen cells from WT mice. In contrast, in cocultures of WT calvarial osteoblasts and spleen cells from NF-kB dKO mice, no TRAP1 cells, either mononuclear or multinucleated were formed.7,12 These in vitvo and in vivo observations indicate that expression of the p50 and p52 subunits of NF-kB is required for osteoclast formation during development and that the defect resides within cells in the osteoclast lineage.
Key Words: NF-kB; Osteopetrosis; Osteoclasts; Lymphocytes; Cytokines.
Address for correspondence and reprints: Brendan F. Boyce, M.D., Department of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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Previous studies of naturally occurring or genetically engineered forms of osteopetrosis have indicated that expression of the transcription factor, PU.1, is required for commitment of precursors to the myelomonocyte lineage29 and that expression of c-fos10,30 and macrophage colony-stimulating factor (MCSF)28,34 is required for commitment of monocyte precursors to the osteoclast lineage. c-fos2/2 and op/op mice form no or very few osteoclasts, but they do produce macrophages, which are present in increased numbers in the marrow cavities of c-fos mutants. Examination of spleen and liver sections revealed that NF-kB dKO mice have normal or increased numbers of macrophages stained using the antimacrophage antibody F4/80.7 Macrophage numbers are also increased in bone marrow,12 indicating that NF-kB functions at a point in the osteoclast lineage very close to that at which c-fos expression is necessary. However, expression of c-fos in the NF-kB dKO mice is normal.12 Thus, both of these transcription factors have critical, nonoverlapping functions in the regulation of genes that commit precursors to the osteoclast lineage at a stage before TRAP is expressed and beyond the point at which monocyte differentiation to macrophages occurs. Since NF-kB regulates the expression of the osteoclastogenic cytokines, interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage (GM)-CSF,5,25 it is possible that osteoclasts fail to form in the NF-kB dKO mice because these cytokines do not get expressed. To determine if expression of these cytokines was normal in NF-kB dKO mice, peritoneal macrophages were elicited from the dKO mice, stimulated with lipopoly saccharide (LPS), and mRNA was extracted. Basal levels of expression of IL-6, IL-1a, and b, and TNF were mostly normal, and expression increased in response to LPS in a manner similar to that seen in WT littermates.7,12 However, expression of IL-6 protein was reduced in dKO macrophages under basal and stimulated conditions.12 Although mRNA expression of these cytokines appears to be normal in peritoneal macrophages from the NF-kB dKO mice, it is possible that production of these proteins by stromal cells or by cells in the osteoclast lineage is defective. To determine if the defect in osteoclast formation could be rescued in vitro by addition of these cytokines, we treated cocultures of osteoblasts and spleen cells from NF-kB dKO mice with IL-1, IL-6, IL-6, and soluble IL-6 receptor (sIL-6R) or GM-CSF alone and in combination. No TRAP1 mononuclear or multinucleated cells were detected in cultures treated with IL-6, IL-1, TNF, or GM-CSF. However, small numbers of TRAP1 multinucleated and mononuclear cells were detected in cultures treated with IL-6 1 sIL-6R.32 Occasional TRAP1 multinucleated cells are also observed in bone sections from NF-kB dKO mice,7 but we do not yet know if these cells or the TRAP1 cells formed in vitro after treatment with IL-6 1 sIL-6R have the capacity to resorb bone. However, if these cells and the multinucleated cells formed when WT osteoblasts and spleen cells from NF-kB dKO mice are treated with IL-6 1 sIL-6R can resorb bone, our findings suggest that some osteoclasts can form in the absence of NF-kB p50 and p52 signaling. We do not know yet if the in vivo mechanism for occasional osteoclast formation is upregulated by IL-6 1 sIL-6 R. It will be important to determine if formation of these TRAP1 cells is mediated by other members of the NF-kB family or if it occurs by an NF-kB-independent mechanism. Recent studies have indicated that osteoclast formation in vitro requires expression of RANK (receptor activator of NF-kB) ligand (RANKL),1 also known as ODF,33 TRANCE,31 and osteoprotegerin (OPG) ligand,17 along with M-CSF by osteoblasts/stromal cells. RANKL binds to its receptor, RANK, on the surface of TRAP1 osteoclast precursors.17 Thus, the failure of osteoclast formation in the NF-kB dKO mice could be due to the
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absence of NF-kB signaling after RANKL/RANK interaction, or because NF-kB dKO mice do not express RANK or c-fms, the tyrosine kinase receptor for M-CSF. c-fms and RANK are expressed in NF-kB dKO mouse spleen cells at levels similar to those in WT mice.32 However, it remains to be shown that the RANK-expressing cells in spleens of NF-kB dKO mice are osteoclast precursors, rather than cells in other lineages, before it can be concluded definitively that the defect is downstream of RANKL/RANK interaction. RANKL has also been shown to stimulate the resorptive activity of isolated osteoclasts in vitro,8,17 suggesting that if RANKL/RANK signaling in these circumstances is mediated through NF-kB, NF-kB may have critical regulatory roles at multiple stages of osteoclast function and activity. NF-kB could also play a significant role in the regulation of the increased osteoclastogenesis associated with estrogen deficiency and inflammatory conditions affecting bone in which expression of the osteoclastogenic cytokines, IL-1, IL-6, and TNF, are increased.11,14,21 NF-kB has been implicated in postmenopausal bone loss because it regulates IL-6 expression through its interaction with the estrogen receptor (ER).9,26 After binding of estrogen to its receptor in osteoblasts, the ER interacts with NF-kB heterodimers after they have been released from the inhibitory binding protein, IkB.9,26 This interaction with the ER prevents NF-kB from binding to response elements in the IL-6 promoter and activating gene expression. Thus, osteoclastogenesis may be kept in check in states of estrogen repletion as a result of this inhibitory effect on IL-6 expression. After the menopause, the inhibitory effect of estrogen and ER interaction with NF-kB is withdrawn, and increased osteoclastogenesis may ensue as a consequence of enhanced IL-6, IL-1, and TNF production by bone marrow macrophages through regulatory loops2,3 in which NF-kB activation leads to enhanced production of these cytokines and more NF-kB activation with subsequent increased production of RANKL and M-CSF by stromal cells. Production of IL-1, IL-6, and TNF by synovial cells and macrophages is increased in patients with rheumatoid arthritis (reviewed in refs. 13 and 27) and may account for the increased osteoclastogenesis and bone loss seen around affected joints in patients with this disorder. NF-kB may play in a role in this increased osteoclastogenesis by activating the expression of these cytokines locally in tissues of affected joints. At present, it is not known which NF-kB proteins might be involved in regulation of the increased osteoclastogenesis in these disorders. NF-kB may also regulate osteoclast survival. Recent studies have indicated that treatment of isolated osteoclasts with inhibitors of NF-kB20 or with antisense oligonucleotides15 to the p65 or p50 subunits of NF-kB induces apoptosis. We have treated osteoclasts generated from bone marrow cultures with the NF-kB inhibitors, TPCK, PDTC, and MG-132, and have found that they promote apoptosis of these cells at doses that have been shown to prevent translocation of NF-kB from the cytoplasm to the nucleus. However, apoptosis of stromal cells was also observed in these cultures at these concentrations and also at significantly lower concentrations. Our previous studies indicate that osteoblast/stromal cell function in the NF-kB dKO mice is normal (they are able to support osteoclast formation from WT spleen cells); bone formation is unimpaired in NF-kB dKO mice and there is no evidence of increased osteoblast apoptosis in bone from the dKO mice (B. F. Boyce, unpublished observation). Thus, the increased osteoclast apoptosis induced by these agents may be nonspecific and mediated through a mechanism that might not involve NF-kB signaling. Finally, NF-kB also appears to have a role in limb development. Limb formation occurs as a result of interaction between mesenchynal and overlying epithelial cells in the apical epider-
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mal ridge in the primitive limb bud (reviewed in ref. 22). Inhibition of NF-kB c-Rel expression in mesenchymal cells results in reduction of limb size and failed development of the bones of the hands and feet, because NF-kB-activated FGF10 production does not occur.6,16 This, however, is a nonessential function of c-Rel because limb development in c-Rel knockout mice is normal. Since the original observation that NF-kB signaling was involved in kappa light chain production,24 many functions have been identified for NF-kB in numerous cell types. So far, essential functions for NF-kB p50 and p52 proteins have been identified for the formation osteoclasts and mature B cells.7 It is possible that these or other members of the NF-kB family of proteins have important, though nonessential, regulatory roles in other bone cell types, which may be revealed by future studies of osteoblasts and chondrocytes from these or other NF-kB knockout animals. References 1. Anderson, D. M., Maraskovsky, E., Billingsley, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179; 1997. 2. Baldwin, A. S., Jr. The NF-kB and IkB proteins: new discoveries and insights. Annu Rev Immunol 14:649 – 83; 1996. 3. Barnes, P. J. and Karin, M. Nuclear factor-kB-a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066 –1071; 1997. 4. Beg, A., Sha, W., Bronson, R., Ghosh, S., and Baltimore, D. Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-kB. Nature 376:167–170; 1995. 5. Beg, A. A. and Baltimore, D. An essential role for NF-kB in preventing TNF-a-induced cell death. Science 274:782–784; 1996. 6. Bushdid, P. B., Brantley, D. M., Yull, F. E., et al. Inhibition of NF-kB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis. Nature 392:615– 618; 1998. 7. Franzoso, G., Carlson, L., Xing, L., et al. Requirement for NF-kB in osteoclast and B cell development. Genes Dev 11:3482–3496; 1997. 8. Fuller, K., Wong, B., Fox, S., Choi, Y., and Chambers, T. TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001; 1998. 9. Galien, R. and Garcia, T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Nucleic Acids Res 25: 2424 –2429; 1997. 10. Grigoriadis, A. E., Wang, Z.-Q., Cecchini, M. G., et al. c-Fos: A key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266:443– 448; 1994. 11. Horowitz, M. C. Cytokines and estrogen in bone; anti-osteoporotic effects. Science 260:626 – 627; 1993. 12. Iotsova, V., Caamano, J., Loy, J., Lewin, A., and Bravo, R. Osteopetrosis in mice lacking NF-kB1 and NF-kB2. Nature Med 3:1285–1289; 1997. 13. Isomaki, P., and Punnonen, J. Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann Med 29:499 –507; 1997. 14. Jilka, R. L. Cytokine, bone remodeling, and estrogen deficiency. J Bone Min Res 23:75– 81; 1998.
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