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Calcified Tissue International DOI: 10.1007/s00223-002-1048-z 2002 Springer-Verlag New York, Inc.

Matrix Proteins versus Cytokines in the Regulation of Osteoblast Function and Bone Formation
M. Horowitz
Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, CT 06510, USA Received: 15 May 2002; Accepted: 10 June 2002; Online publication: 21 October 2002 Correspondence to: M. Horowitz Email: mark.horowitz@yale.edu

As my counterpart (http://link.springer.de/link/service/journals/00223/contents/02/1017/paper/index.html) has mentioned it is true, there is a lot of matrix. However, I would not argue that because there is so much it is not important. Quite the contrary, there is so much matrix because it is needed to carry out its primary functions as the precursor to bone and the major store for calcium and phosphate. I will demonstrate the importance of cytokines and growth factors in the regulation of osteoblast function and bone formation by selecting examples that illustrate different aspects of cytokine-mediated regulation of bone metabolism. As mentioned in the previous section (http://link.springer.de/link/service/journals/00223/contents/02/1017/paper/index.html), cytokines are present in much smaller quantities in skeletal tissue as compared with matrix proteins. This is the nature of these factors due to their potency. Cytokines are small polypeptide molecules that bind to specific high-affinity cell surface receptors. Constitutive production is usually low or absent. They are produced by cells locally, usually have a short action radius (autocrine or paracrine not endocrine), and function transiently in response to exogenous stimulation. Cytokines are secreted, they deliver their signal, and by design they are gone. Many cytokine mRNA's have specific sequences in the 3 untranslated region, which facilitates rapid degradation. Because of bone's propensity to be regulated at specific anatomic sites, coupled with the local production and tight functional area, cytokines can have major effects on the skeleton. In the adult years, bone resorption is balanced by equivalent bone formation, resulting in maintenance of skeletal mass. This balance is tightly regulated by a number of factors including hormones, physical force, and cytokines. However, it is more than only the induction of formation that maintains bone homeostasis; it is also the inhibition of resorption. In fact, the bone remodeling cycle is never truly at rest, rather, it is actively engaged, being driven by bone-resorbing osteoclasts and remaining under control because of suppressive factors. This was most clearly demonstrated in osteoprotegerin (OPG)-deficient mice. OPG is a naturally circulating member of the TNF receptor gene superfamily [1, 2]. OPG functions as a decoy receptor binding RANKL, which is expressed on stromal and osteoblastic cells. RANKL is a member of the TNF family of

cytokines [3, 4]. This interaction prevents RANKL from interacting with its receptor RANK, which is expressed on osteoclast precursors, resulting in inhibition of the terminal differentiation of osteoclasts and the activation of mature cells. Loss of OPG results in a phenotype with a marked loss in total bone density characterized by obvious trabecular and cortical bone porosity, serious thinning of the parietal bones of the skull, and increased fractures [5]. Conversely, systemic administration or overexpression of OPG results in a dramatic increase in trabecular bone in both vertebra and long bone [6]. This suppression of osteoclast formation is similar, in broad terms, to the suppressive activity estrogen exerts on the skeleton. Thus, OPG is a soluble, circulating cytokine required to prevent run-away bone resorption and maintain the balance between resorption and formation. Although cytokines and growth factors are most often thought of as soluble mediators, this is not always the case. A good example of this is macrophage colony-stimulating factor (M-CSF or CSF-1) which is required at multiple points in the lineage development of osteoclasts. M-CSF is required at the very early stages of osteoclast progenitor differentiation, at the formation of multinucleated functionally active cells, and possibly for the survival of those already formed multinucleated cells [7]. It may be that the expression of the receptor for M-CSF, c-fms a receptor tyrosine kinase, is the event required for osteoclast lineage development. M-CSF has two biologically active forms: a soluble form and a cell surface or membrane-bound form. The cell surface form is the product of alternatively spliced mRNA [8]. Cell surface-expressed M-CSF is sufficient to stimulate osteoclast formation and macrophage proliferation in vitro [9]. Thus, c-fms can bind and be activated by the soluble molecule as well as when M-CSF is presented on the cell surface. This presentation has a number of advantages over the soluble form. First, less of the cell surface form has to be produced. Second, the specificity increases using the cell surface form. M-CSF is expressed on stromal cells and osteoblasts. Cell surface-expressed M-CSF in conjunction with RANKL interact with their respective receptors on osteoclast precursors, resulting in the formation of bone-resorbing osteoclasts. RANKL is functional as a cell surface-expressed molecule or can be cleaved, and functions as a soluble cytokine. This cell-cell interaction explains the long-standing observation that cell contact between stromal cells or osteoblasts and osteoclast precursors is required for osteoclast differentiation in vitro. Therefore, MCSF and RANKL can function as cell-surfaced expressed cytokines that regulate bone cell activity. Cell-cell interactions are also important in osteoblast activation and bone formation. GATA-1 is a zinc finger transcription factor required for megakaryocyte differentiation [10]. GATA-1-deficient mice have a developmental arrest of megakaryocyte differentiation, having only 15% of the normal number of platelets, which fail to function. Interestingly, these mice have a bone phenotype characterized by 34 times the amount of trabecular bone, a 23-fold increase in cortical bone, with a dramatic increase in the number of osteoblasts [11]. The number of osteoclasts in these mice is also increased and they resorb bone, indicating that the increased bone is not due to defective osteoclasts. Osteoblast proliferation is stimulated by GATA-1-deficient megakaryocytes. This stimulation is not mediated by soluble factors but requires contact between GATA-1-deficient megakaryocytes and osteoblasts [12]. Thus, GATA-1 mutant megakaryocytes can deliver a cell surface-derived signal that increases osteoblast number and function.

The fact that target cells can be activated by cell surface-expressed cytokines or growth factors indicates that presentation is important for ligand-receptor interaction. Then it could be argued that the sequestration of TGF in bone is another form of presentation. TGF could be released from bone by osteoclastic activity either enzymatically or by acidification. The release of TGF from bone would be advantageous in situations such as fracture repair in which TGF could play a dual function, first to suppress the inflammatory response which occurs directly after the fracture and second to speed healing by stimulating osteoblast proliferation. However, there is little data to suggest that osteoclastic resorption leads to the release of active TGF, which is functional in situ. Unlike cytokines, the fact that certain matrix proteins (e.g., heparan sulfhate proteoglycans) can bind other proteins, including cytokines and growth factors, does not mean those matrix protein-factor complexes can then engage their receptors and transduce a signal. Alternatively, osteopontin (OPN) functions as both a soluble cytokine and a matrix protein. In bone, OPN is an abundant noncollagenous protein produced by osteoclasts, osteoblasts, and osteocytes. Matrix-bound OPN can bind cells through its GRGDs sequences. This allows osteoclasts to bind to matrix by an OPNv3 integrin interaction. This may be important in estrogen deficiency because OPNdeficient mice are resistant to ovariectomy [13]. OPN is also known as early T lymphocyte activation-1 (Eta-1/Op), a T lymphocyte-derived factor. Secreted OPN appears to play an important role in immune resistance to certain bacterial and viral infections [14]. Lymphocytes and other hematopoietic cells express the CD44 receptor, which regulates homing and attachment [14]. OPN/Eta-1 is a ligand for CD44 accounting for its multiple functions [15]. The data are unequivocal, showing that cytokines regulate osteoblast function, bone formation, and numerous other bone cell activities. I would agree that matrix is not just a scaffold for cells. In fact, bone matrix provides the growth milieu on and in which lining cells, osteoblasts, and osteocytes depend for growth and function. Bone cells thrive because they adhere to matrix proteins, which support their growth. It is possible that cytokines and growth factors embedded in the matrix contribute to their survival. Although I believe that the matrix can regulate bone and other cells, there is little direct evidence to support the idea that this is accomplished through the presentation of cytokines and growth factors. I agree with my counterpart (http://link.springer.de/link/service/journals/00223/contents/02/1017/paper/index.html) that specific cytokines and growth factors in association with matrix can be considered as a functional unit. How the components contribute to the unit is unclear. The matrix, like cytokines, has important functions many of which are independent of each other.

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