JBJS | New Horizons in Orthopaedic Research: Elucidation of Cellular Signal Transduction Pathways

The Journal of Bone & Joint Surgery, Volume 81, Issue 5

Editorial | May 01, 1999

New Horizons in Orthopaedic Research: Elucidation of Cellular Signal Transduction Pathways

KENNETH A ROEBUCK, Ph.D.; JOSHUA J. JACOBS, M.D.; TIBOR T. GLANT, M.D., Ph.D.

The Journal of Bone & Joint Surgery. 1999; 81:599-602

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Introduction

Introduction | References

The report by Nakashima et al.⁶ in this issue of The Journal and the one by Blaine et al.³ in the October 1997 issue dealt with signal transduction mechanisms in macrophages stimulated by orthopaedic biomaterials. Although these pioneering studies represent initial findings and focus on only a narrow range of possible signaling events, they open new vistas within clinically oriented orthopaedic research. The ultimate aim of this new line of research is to delineate the fundamental cellular mechanisms of periprosthetic osteolysis induced by particulate wear debris and to establish strategies for the development of therapeutic agents to prevent, reverse, or retard this adverse outcome of joint replacement procedures. Furthermore, the study of cellular signal transduction pathways can be applied to numerous areas of orthopaedic research, including the growth and development of cartilage and bone, the pathogenesis of osteoarthritis, fracture-healing, and tumor growth, to name but a few.

One of the most important advances in biomedical science in this decade is the identification of intracellular signaling events as part of cellular adaptive responses. These regulatory events are important for mammalian cells to deal with changes in their microenvironment and maintain homeostasis. Cells receive external signals by way of surface membrane receptors, and as a consequence certain amino acids (usually tyrosine, serine, or threonine residues) of the cytoplasmic tail of the receptor become phosphorylated. This initial biochemical event is amplified through a series of protein kinase phosphorylations that in the end trigger a defined set of intracellular adaptive processes, including long-term gene responses. Extracellular compounds in the form of lipids, hormones, growth factors, cytokines, chemokines, counter receptors of other cells, or other cellular mediators can bind to specific cell-surface receptors. These interactions trigger intracellular signal-transduction cascades that result in the modulation of nuclear transcription factor activity. Transcription factors are nuclear sequence-specific DNA-binding proteins that activate gene transcription by interacting with the promoter regions of responsive genes. Multiple signaling pathways activated by different receptors may converge and interact, resulting in a highly specific cell response. In contrast, cell activation by a single receptor may diverge and activate various signaling pathways with diverse cellular responses. For example, the proinflammatory cytokines IL-1 (interleukin-1), TNF-a (tumor necrosis factor-alpha) and IL-6 (interleukin-6) activate a network of signal transduction pathways leading to a well defined inflammatory response (Fig. 1). These cytokines are regulated at both the transcriptional and the post-transcriptional level. For example, when IL-1 is bound to its own receptor, it stimulates the secretion of IL-1 from that cell. Simultaneously, the genes for the cytokines TNF-a and IL-6, which contain cytokine responsive elements within their promoter regions, become transcriptionally active, synthesizing new mRNA (messenger RNA) and protein. These newly synthesized cytokines are secreted from the cell to bind their receptors either on the same cell (an autocrine effect) or on other types of cells (a paracrine effect) and further amplify the cytokine-mediated response. Unchecked production of cytokines can lead to persistent stimulation of the inflammatory response, causing chronic inflammation and tissue damage such as that associated with periprosthetic osteolysis.

The genes for IL-1, THF-a, and IL-6 are regulated by inducible transcription factors, which are activated through signaling pathways involving tyrosine phosphorylation cascades. The trnascription factors NF-?B (nuclear factor-kappa B) and NF-IL-6 (nuclear factor-IL-6) and two different families of transcription factors containing multiple members that bind to their DNA sites as functional dimers. In resting cells, NF-?, usually composed of the nucleus, all of which involve protein phosphorylation (Fig. 1). The three signaling mechanisms are exemplified by the transcription factors NF-IL-6, NF-?B, and STAT-3. The first mechanism depends on the regulated translocation of activated protein kinases from the cytoplasm to the nucleus, where they phosphorylate target transcription factors (NF-IL-6). In the second mechanism, transcription factors are kept in a latent state in the cytoplasm and are translocated into the nucleus on activation (NF-?B). The last mechanism involves the release of transcription factors from cytoplasmic proteins. The monomeric transcription factors then form dimers and translocate to the nucleus (STAT-3).

As reported by Nakashima et al.⁶ in this issue and by Blaine et al.³, particulate wear debris can initiate signal transduction pathways in monocytes and macrophages, leading to the production and release of TNF-a and IL-6. Although the mechanisms by which particles activate macrophages have not been fully explored, induction of cytokine genes was accompanied by increases in tyrosine kinase activity and activation of NF-?B and NF-IL-6, both of which have been shown to be important for the transcriptional activation of the TNF-a and IL-6 genes. NF-IL-6 and NF-?B can physically interact and together can functionally cooperate to activate gene transcription^9,10. Cytochalasin B, which inhibits cytoskeleton reorganization and tubulin polymerization, is routinely used to inhibit phagocytosis. However, neither cytochalasin B nor its more potent cousin, cytochalasin D, is a perfect inhibitor of phagocytosis, even though both compounds can markedly reduce the rate of phagocytosis and the number of engulfed particles. Nakashima et al.⁶ showed that less than 10 percent of macrophages had phagocytosed particles at the one-hour time-point with use of optimum concentrations of cytochalasin B. However, these concentrations, which preserve cell viability, do not completely block the phagocytosis. In addition, nonphagocytosed particles cannot be completely removed from the culture system even after trypsinization and centrifugation. Thus, even in a well defined cell-culture system, phagocytosis still occurs and tends to increase with time, so that the levels of cytokines measured in the medium at twenty-four or forty-eight hours may reflect, to some extent, this low residual level of phagocytosis.

Nevertheless, Nakashima et al.⁶ showed that, even in the setting of markedly reduced phagocytosis, an initial signal can be generated by a particle-bound ligand, which may be sufficient to trigger signaling cascades in monocytes and macrophages. Although those authors concluded that release of IL-6 and TNF-a from macrophages was independent of phagocytosis, and alternative hypothesis, consistent with their data, is that soluble compounds (for example, ß1 integrins, complement, and Fc-containing immunoglobulins) bound to the particle surface (opsonized) may transmit a sufficient signal for an initial cell activation followed by either normal or so-called frustrated phagocytosis. Interestingly, large (nonphagocytosable) particles in similar experimental setups, with use of fibroblasts and osteoblasts, remain inactive^13,14, which suggests that particle size might be a critical parameter in cell activation. This finding supports the potential role of phagocytosis in the observed cell response. Moreover, because the serum used in the study by Nakashima et al.⁶ was not heat-inactivated, the possibility that the cell response may be, at least in part, a consequence of soluble complement factors (such as C3b) in the serum, as opposed to particle-bound component factors, cannot be excluded. Clearly, additional studies are necessary to determine the molecular basis for the particle-mediated signaling and the role of phagocytosis as an initiating or potentiating event.

In another related study in this issue of The Journal, Wooley et al.¹² analyzed the surface-bound proteins on polyethylene components retrieved from patients who failed joint replacement. A total of twenty-one proteins resolved by polyacrylamide gel electrophoresis were isolated from these components, including type-I colagen and immunoglobulin in most explants. In addition, the authors detected autoantibodies to various matrix molecules, mostly against type-I collagen, in the sera of most of these patients. The authors hypothesized that polyethylene-bound immunoglobulins, complexed to matrix protein antigens, may fix complement and initiate the complement cascade, thereby attracting inflammatory cells to the vicinity. They went on to speculate that polyethylene in particulate form could provide an adjuvent effect for the response to polyethylene-bound proteins. Although these observations are very interesting, they beg the question of whether any of these patients had had autoantibodies before the joint replacement or whether these antibodies appeard de novo after the arthroplasty. It must be noted that all patients already had a defect in the musculoskeletal system (for example, osteoarthritis or osteonecrosis), and possibly in the immune system as well (for example, rheumatoid arthritis or one of its variants), before the joint replacement because progressive destruction of the joint was the indication for the arthroplasty. In addition, the prevalence of polyethylene-bound proteins and serum matrix-protein autoantibodies in patients who had well functioning, clinically successful devices is not known, so the association of these factors with failure of the implant and periprosthetic inflammation is incomplete. In any event, this is a provocative study that suggests a new pathway for the development of the periprosthetic inflammatory response.

Taken together, the studies by Nakashima et al.⁶ and Wooley et al.¹² underscore the importance of surface interactions in the biological process of aseptic loosening. In one study, the interaction between particles and the cell surface was hypothesized to promote the activation of various intracellular signaling pathways leading to the elaboration of proinflammatory cytokines. In the other study, biomaterial surface-bound proteins were hypothesized to be an important component in the initiation and maintenance of the inflammatory response to particulate wear debris.

In conclusion, particulate wear debris can set up a catastrophic inflammatory reaction involving the activation of a number of cell types, including macrophages^2,3,5,6, fibroblasts¹³, osteoblasts^4,14, and osteoclasts¹¹. The key players in this pathological process appear to be the inflammatory transcription factors NF-?B and NF-IL-6, which activate a set of proinflammatory mediators, including IL-1, THF-a and IL-6. These cytokines fuel the vicious cycle of chronic inflammation leading to deterioration of the local osseous architecture and eventually to periprosthetic osteolysis and aseptic loosening. Future studies will focus on understanding the intracellular signaling events in this inflammatory disease process in the hope of developing effective strategies and identifying critical targets suitable for therapeutic intervention.

Kenneth A. Roebuck, Ph.D.

Joshua J. Jacobs, M.D.

Tibor T. Glant, M.D., Ph.D.

Rush University at Rush-Presbyterian-St. Luke's Medical Center

Chicago, Illinois

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Fig. 1 Schematic representation showing the inflammatory signal transduction network. The proinflammatory cytokines TNF-a, IL-1ß, and IL-6 activate three general signal transduction pathways exemplified by the activation of the nuclear transcription factors NF-?B, NF-IL-6, and STAT-3. TNF-a interacts with the THF-a receptor (TNFR), setting off a cascade of protein phosphorylation leading to the activation of the NF-?B-inducible kinase (NIK), which in turn activates the I-?B kinase (IKK). IKK phosphorylates the inhibitor protein I-?B, releasing NF-?B (p65/p60 heterodimer) to translocate to the nucleus, and binds its recognition site in the promoter regions of the TNF-a and IL-6 genes. IL-1ß interacts with the IL-1ß receptor (IL-1R) and activates NF-?B and the mitogen activation protein kinase (MAPK) pathway (Ras-Raf-MEK). This activation leads to the phosphorylation of NF-IL-6, which binds to the TNF-a and IL-6 promoter cooperatively with NF-?B. Together, they recruit coactivator proteins and activate TNF-a and IL-6 gene transcription. TNF-a and IL-6 can be secreted from the cell and have secondary autocrine and paracrine effects on nearby cells. IL-6 binds its receptor (IL-6R) to activate the JAK/STAT (Janus activating tyrosine kinase/signal transduction and activator of transcription) pathway. In response to activation of the IL-6R, JAK phosphorylates STAT-3, which allows it to form homodimers, translocate to the nucleus, bind IL-6 responsive elements (IL-6RE) within the NF-IL-6 promoter, and activate NF-IL-6 gene transcription. Newly synthesized NF-IL-6 protein then can provide feedback and activate the late expression of the TNF-a and IL-6 genes by interacting with their promoters.

References

Introduction | References

Baldwin, A.S., Jr.: The NF-kappa B and I kappa B proteins: new discoveries and insights. Ann. Rev. Immunol.,14: 649-683, 1996.14649 1996

Blaine, T.A.; Rosier, R.N.; Puzas, J.E.; Looney, R.J.; Reynolds, P.R.; Reynolds, S.D.; and O'Keefe, R.J.: Increased levels of tumor necrosis factor-a and interleukin-6 protein and messenger RNA in human peripheral blood monocytes due to titanium particles. J. Bone and Joint Surg.,78-A: 1181-1192, Aug. 1996.78-A1181 1996

Blaine, T.A.; Pollice, P.F.; Rosier, R.N.; Reynolds, P.R.; Puzas, J.E.; and O'Keefe, R.J.: Modulation of the production of cytokines in titanium-stimulated human peripheral blood monocytes by pharmacological agents. The role of cAMP-mediated signaling mechanisms. J. Bone and Joint Surg.,79-A: 1519-1528, Oct. 1997.79-A1519 1997

Glant, T.T.; Dobai, J.G.; Ramesh, N.; Chandrasekaren, R.; Carpenter, L.; Galante, J.O.; and Roebuck, K.: Phagocytosis of titanium particulates activates protein tyrosine kinase pathway and transcription factor NF-?B, upregulates IL-6 production and suppresses collagen synthesis in osteoblasts. Trans. Orthop. Res. Soc.,24: 246, 1999.24246 1999

Lee, S.-H.; Brennan, F.R.; Jacobs, J.J.; Urban, R.M.; Ragasa, D.R.; and Glant, T.T.: Human monocyte/macrophage response to cobalt-chromium corrosion products and titanium particles in patients with total joint replacements. J. Orthop. Res.,15: 40-49, 1997.1540 1997 [PubMed]

Nakashima, Y.; Sun, D.-H.; Trindade, M.C.D.; Maloney, W.J.; Goodman, S.B.; Schurman, D.J.; and Smith, R.L.: Signaling pathways for tumor necrosis factor-a and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro. J. Bone and Joint Surg.,81-A: 603-615, May 1999.81-A603 1999

Poli, V.: The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem.,273: 29279-29282, 1998.27329279 1998 [PubMed]

Roebuck, K.A.; Carpenter, L.R.; Lakshminarayanan, V.; Page, S.M.; Moy, J.N.; and Thomas, L.L.: Stimulus-specific regulation of chemokine expression involves differential activation of the redox-responsive transcription factors AP-1 and NF-?B. J. Leukocyte Biol.,65: 291-298, 1999.65291 1999 [PubMed]

Stein, B.; Cogswell, P.C.; and Baldwin, A.S., Jr.: Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Molec. and Cell. Biol.,13: 3964-3974, 1993.133964 1993

Stein, B., and Baldwin, A.S., Jr.: Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. Molec. and Cell. Biol.,13: 7191-7198, 1993.137191 1993

Wang, W.; Ferguson, D.J.; Quinn, J.M.; Simpson, A.H.; and Athanasou, N.A.: Osteoclasts are capable of particle phagocytosis and bone resorption. J. Pathol.,182: 92-98, 1997.18292 1997 [PubMed]

Wooley, P.H.; Fitzgerald, R.H., Jr.; Song, Z.; Davis, P.; Whalen, J.D.; Trumble, S.; and Nasser, S.: Proteins bound to polyethylene components in patients who have aseptic loosening after total joint arthroplasty. A preliminary report. J. Bone and Joint Surg.,81-A: 616-623, May 1999.81-A616 1999

Yao, J.; Glant, T.T.; Lark, M.W.; Mikecz, K.; Jacobs, J.J.; Hutchinson, N.I.; Hoerrner, L.A.; Kuettner, K.E.; and Galante, J.O.: The potential role of fibroblasts in periprosthetic osteolysis: fibroblast response to titanium particles. J. Bone and Min. Res.,10: 1417-1427, 1995.101417 1995

Yao, J.; Cs-Szabó, G.; Jacobs, J.J.; Kuettner, K.E.; and Glant, T.T.: Supression of osteoblast function by titanium particles. J. Bone and Joint Surg.,79-A: 107-112, Jan. 1997.79-A107 1997

Topics

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Add to My JBJS

Baldwin, A.S., Jr.: The NF-kappa B and I kappa B proteins: new discoveries and insights. Ann. Rev. Immunol.,14: 649-683, 1996.14649 1996

Poli, V.: The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem.,273: 29279-29282, 1998.27329279 1998 [PubMed]

Wang, W.; Ferguson, D.J.; Quinn, J.M.; Simpson, A.H.; and Athanasou, N.A.: Osteoclasts are capable of particle phagocytosis and bone resorption. J. Pathol.,182: 92-98, 1997.18292 1997 [PubMed]

Yao, J.; Cs-Szabó, G.; Jacobs, J.J.; Kuettner, K.E.; and Glant, T.T.: Supression of osteoblast function by titanium particles. J. Bone and Joint Surg.,79-A: 107-112, Jan. 1997.79-A107 1997

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These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.

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KENNETH A ROEBUCK, JOSHUA J. JACOBS, TIBOR T. GLANT; New Horizons in Orthopaedic Research: Elucidation of Cellular Signal Transduction Pathways. The Journal of Bone & Joint Surgery. 1999 May;81(5):599-602.

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