Bone Cell Precursors and the Pathophysiology of Bone Loss

Bone Cell Precursors and the Pathophysiology of Bone Loss HARRY C. BLAIR, a AND JILL L. CARRINGTON b a Departments of Pathology and Cell Biology, University of Pittsburgh, and Pittsburgh VA Medical Center, Pittsburgh, Pennsylvania 15213, USA b Biology of Aging Program, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892, USA ABSTRACT: In health, changes in bone formation and degradation rates are coupled and adequate cellular resources are available in the bone so that a change in bone formation rate occurs with an opposing change in resorption. On the other hand, the regulation of bone volume, particularly in pathological conditions, is dependent not only on the pathways that mediate terminal pathways of bone cell differentiation, but also on the availability of stem cells for allowing the differentiation to occur. Regulation of cell numbers in stem cell compartments and release of stem cells for differentiation of osteoblast or osteoclast precursors are not well understood, although it is clear that changes in stem cell numbers underlie pathological changes in bone mass. This may include effects of aging, fracture, metastatic disease, and autoimmune diseases on the precursor cell pools available for bone formation and degradation. Increases in osteoclast precursors or decreases in osteoblast precursors are common features of bone-losing states; increases in precursors may conversely occur during growth or repair processes. Rational therapy based on modifying stem cell populations may, when the processes are better understood, help prevent chronic bone-losing states and may also be of use in preventing or treating aplastic anemia and related conditions. KEYWORDS: stem cells; osteoblast; osteoclast; mesenchymal cells; aplastic anemia; autoimmunity; differentiation MARROW MESENCHYMAL AND HEMATOPOIETIC STEM CELLS Bone matrix is derived from mesenchymal stem cells that can produce chondrocytes and osteoblasts or which may undergo alternative differentiation to vascular, adipose, connective, and muscular tissue. These cells can be isolated Address for correspondence: Harry C. Blair, 705 Scaife Hall, University of Pittsburgh, Pittsburgh, PA 15261. Voice: 412-383-9616; fax: 412-647-8567. e-mail: hcblair@imap.pitt.edu Ann. N.Y. Acad. Sci. 1068: 244 249 (2006). C 2006 New York Academy of Sciences. doi: 10.1196/annals.1346.028 244

BLAIR & CARRINGTON: STEM CELLS AND BONE LOSS 245 from marrow 1 and are generally regarded as the durable, self-replicating pool of adherent bone cell precursors for turnover and repair. Bone degradation requires vascular endothelial growth factor (VEGF) and vascular ingrowth, 2 this is generally regarded as necessary for supplying marrow cell precursors to enter into bone, which ultimately will produce circulating monocytic cells that further differentiate to osteoclasts. 3 The relation between the most primitive mesenchymal cell precursors and the least differentiated hematological stem cells is uncertain, but recent work suggests that parathyroid hormone related protein (PTHrP) or parathyroid hormone (PTH) influence factors including the Notch ligand jagged-1, 4 which supports early hematopoietic precursor proliferation, and helps explain earlier observations pointing to the production of other early myeloid growth factors including stem cell factor, 5 by osteoblasts and osteoblast-related mesenchymal cells. Thus, an important auxiliary function of bone is maintenance of the self-renewing marrow stem cell populations that are required for lifelong bone turnover and also for hematopoiesis. 6 The relationship of marrow stem cell failure in aplastic anemia with mesenchymal cell growth factors is a poorly defined function of great importance, but is not further considered here, where we will concentrate on the more distal osteoclast and osteoblast precursor populations. The bone marrow contains mononuclear-precursor and mesenchymal stem cells capable, respectively, of producing CD11b osteoclast precursors 7 and committed osteoblasts. 1 There are some indications that there may be earlier primitive monocyte-like stem cells capable of replacing both hematopoietic and mesenchymal stem cells, although these may also represent separate populations of small monocyte-like antigen negative null cells. For example, nonadherent null monocytic cells replace both osteoblasts and marrow elements after lethal irradiation. 8 The issue as to whether this represents the effects of broadly pluripotent cells or multiple committed stem cells of different lineages has been difficult to resolve due to the lack of distinguishing antigens on the cells and to lack of understanding of conditions for the maintenance and differentiation of the earliest stem cells of either the hematopoietic or mesenchymal stem cell components. However, common clonal origin of osteoblastic and marrow cells in mice 8 suggests that a limited number of such pluripotent cells capable of producing mesenchymal stem cells and hematopoietic stem cells may exist in some adult mammals. CIRCULATING STEM CELLS Parabiotic experiments with green fluorescent protein mice have established that stem cells, which are abundant in specialized environments such as the marrow, also circulate in appreciable numbers and can regenerate cells in several mesenchymal tissues including muscle. 9 There are also more mature adherent cells that function well as osteoblastic precursors. 10 Kuznetsov et al. 11

246 ANNALS NEW YORK ACADEMY OF SCIENCES reported that these cells are very rare in humans, on the order of one in 10 8 circulating nucleated cells, calling into question their physiological relevance in most cases. On the other hand, there are no specific data as to earlier mesenchymal/osteoblast precursors that may not be captured by the analysis of adherent cells. Eghbali-Fatourechi et al. have addressed this issue of the circulating committed osteoblastic precursor in humans by analyzing osteocalcin expressing, or BAP expressing, nonadherent monocytic circulating cells in adolescent and adult humans and after fracture. This recently published work 12 suggests that osteocalcin-expressing CD15 negative cells circulate in quantity, and may represent nearly 1% of monocytic cells in healthy adults and even higher circulating quantities during rapid growth or after fracture. This work, discussed in detail by Khosla in a subsequent paper in this volume, suggests that a circulating pool of nonadherent cells is an important mechanism in repair and possibly also during normal modeling. Problems still to be resolved include determination of whether some or all of the sorted osteocalcin expressing cells are capable of bone formation, and the specific signals required to cause localization and differentiation of the circulating cells to skeletal sites where osteoblast precursors are required. This work may also help explain the variation in primitive monocytic cells detected on automated screening of leukocytes in blood, which has long been a problem for clinicians, and suggests that in the case of questionable results that sensitive analysis for the early osteoblast product osteocalcin may differentiate between true pluripotent blasts versus dedicated bone cell precursors. REGULATION OF BONE PRECURSORS BY TUMOR CELLS Osteolytic and sclerotic lesions occur in varying frequency with different types of cancer. The most common tumors producing bone metastasis are prostate or breast cancer and multiple myeloma, with prostate cancer characteristically producing sclerotic or osteoblastic lesions, while breast cancer and myeloma typically produce lytic lesions. Of the several types of metastatic tumors leading to osteolytic lesions, myeloma is one of the most problematic as well as the most consistent tumor type, with characteristically punch out lesions that are almost purely osteolytic. Specific mechanisms center around the production of growth factors by tumor cells; these include PTHrP and several interleukins as well as TNF-family proteins and tyrosine kinase agonists including colony stimulating factor-1 (CSF-1). 13,14 In addition, tumor cells produce immune cell response via multiple mechanisms and result in secondary changes including stromal proliferation, and vascular growth factors that can contribute to osteolytic mechanisms. Specific tumor cytokines produce additional dramatic specific effects on bone loss, as is most well characterized in myeloma by the work of Abe et al. 15

BLAIR & CARRINGTON: STEM CELLS AND BONE LOSS 247 and independently by Choi et al., 16 who found that the metastatic myeloma cells produce high levels of macrophage inflammatory peptide-1 (MIP-1) alpha or beta. This subject is reviewed in detail by Matsumoto in a subsequent paper in this volume. In brief, antibodies to MIP-1 or its receptor CCR5 greatly diminish the effects of myeloma supernatants on osteoclast formation, validating this as a key mechanism. Mechanisms that are involved include direct RANKLbased mechanisms, tumor cell adhesion effects via VLA4/VCAM, production of VEGF by tumor cells, production by bone cells of growth factors including IL-6 that foster tumor survival, and suppression of bone formation by tumor production of wnt-related inhibitors including sfrp-2. There are reports of other wnt-related proteins in myeloma cells including Dkk1, 17 although the sfrp-2 mechanism appears to be more commonly expressed by this tumor type. IMMUNITY, AUTOIMMUNITY, AND BONE LOSS REFLECT CHANGES IN BONE CELL PRECURSOR POOLS Aside from the view that gonadal failure produces a pseudo-autoimmune state, as particularly described 18 and discussed by Pacifici elsewhere in this volume, there are substantive data related both to autoimmune states and bone loss and to secondary bone loss related to therapy-targeting autoimmune states. In some cases, such as rheumatoid arthritis, the predominant mechanism is related to the effects of the key cytokine tumor necrosis factor- (TNF- ), which has both direct and indirect effects on bone and joint cell survival via effects on macrophages and T lymphocytes with potential downstream effects on bone and joint destruction via macrophages, chondroclasts, and osteoclasts. 19 Understanding of autoimmune and rheumatological states clinically is complicated by the uniformly unfavorable effects of immunosuppressive agents, either in the corticosteroid or more specific calmodulin-inhibitor families, on bone mass. Nonetheless, it is clear that numerous autoimmune or rheumatological states result in a direct deleterious effect on bone mass by more indirect mechanisms. A key indirect mechanism related to bone loss in autoimmune disorders is modification of the precursor pool for the formation of bone and jointdegrading cells. As is considered in detail by Schwartz later in this volume, studies of autoimmune polyarthritis, which is associated with TNF- overexpression, relative to systemic lupus erythematosus, in which interferon production is predominant, reveal that in disease with mainly TNF- overexpression, there is a specific increase in CD11b/CD14 pools of osteoclast precursors that enhance osteoclastic response. 20 In diseases such as systemic lupus erythematosus (SLE), with elevated interferon-, CD11b pools are depleted in differentiation toward CD11c-expressing dendritic cells, 21 the autoimmune diseases do not have a natural bone- or joint-destructive phenotype but rather

248 ANNALS NEW YORK ACADEMY OF SCIENCES are subject to bone loss, largely in response to the anti-inflammatory therapy used to manage the autoimmune disease. The cells involved may be quite early in the macrophage-monocyte precursor series, and may be Fms-positive or -negative and RANK-positive or -negative. These mechanisms, it should be emphasized, being relatively early in the differentiation process of bone cells, affect the precursor cell populations, an entirely separate issue from the late effects of TNF- on osteoclastic differentiation, which have been described by several workers in vitro. 22,23 Effects of TNF family proteins other than RANKL in osteoclast differentiation are complex, and may have both proand antiresorptive effects depending on cell maturation, time of exposure, and microenvironmental factors. ACKNOWLEDGMENTS This research was supported by the New York Academy of Sciences, by the Department of Veteran s Affairs, and by National Institutes of Health (USA) grants AR47700 and AG12951. REFERENCES 1. PITTENGER, M.F. et al. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284: 143 147. 2. ENGSIG, M.T. et al. 2000. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J. Cell Biol. 151: 879 889. 3. FUJIKAWA,Y.et al. 1996. The human osteoclast precursor circulates in the monocyte fraction. Endocrinology 137: 4058 4060. 4. CALVI, L.M. et al. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425: 841 846. 5. BLAIR, H.C. et al. 1999. Parathyroid hormone-regulated production of stem cell factor in human osteoblasts and osteoblast-like cells. Biochem. Biophys. Res. Commun. 255: 778 784. 6. VISNJIC, D. et al. 2004. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 103: 3258 3264. 7. FERRON, M.& J. VACHER. 2005. Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice. Genesis 41: 138 145. 8. DOMINICI, M. et al. 2004. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc. Natl. Acad. Sci. USA 101: 11761 11766. 9. SHERWOOD, R.I. et al. 2004. Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem Cells 22: 1292 1304. 10. LONG, M.W. et al. 1995. Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors. J. Clin. Invest. 95: 881 887. 11. KUZNETSOV, S.A. et al. 2001. Circulating skeletal stem cells. J. Cell Biol. 153: 1133 1140.

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