In this study, we demonstrated that PTH is capable of increasing bone mass in myelomatous bones in vivo
and that the increased bone formation is associated with a concomitant reduction in growth of the Hg myeloma cell line and primary myeloma cells from certain patients. In our animal model, pretreatment with PTH also resulted in increased bone mass and a significant delay in MM progression. Treatment with PTH markedly increased the number of differentiating osteoblasts, but the number of osteoclasts remained unchanged in bones engrafted with Hg myeloma cells and was moderately reduced in bones engrafted with primary myeloma cells. Strongly supporting our findings, GEP analyses of whole myelomatous bones showed increased expression of osteoblastic markers and reduced expression of osteoclastic and myeloma cell markers. GEP analyses also provided insight on molecular mechanisms that mediate the various effects of PTH in myelomatous bones. Because PTH had no direct effects on growth of myeloma cells, we conclude that shifting bone turnover to an anabolic state in myelomatous bone results in negative effects on MM progression. The results of this study support our previous findings, and those of others, that increased bone mass resulting from exogenous MSC cytotherapy 
or treatment with DKK1-neutralizing antibody 
, Wnt3a 
, or lithium chloride 
negatively impact MM tumor burden in bone.
PTH is approved for treatment of osteoporosis in men and women 
, but patients with cancer currently are not treated with PTH because of concerns that the treatment might promote tumor growth or osteosarcoma 
. In the present study, we tested the effect of a relatively high dose of PTH (80 µg/kg/d) on MM bone disease and tumor growth in our animal models. Similar high doses had previously been tested in animal models for osteoporosis 
. Although analogy to the clinical setting cannot properly be made due to the significantly higher metabolic rate of mice compared to humans, it is of interest to test whether lower doses of PTH have a significant effect on prevention of MM bone disease. Our study demonstrated not only that PTH has no direct stimulatory effects on myeloma cells but also, intriguingly, that PTH has antitumor properties, presumably due to its ability to alter the bone marrow microenvironment. Although PTH has been shown to promote osteoclastogenesis in certain (but not all) physiological and experimental conditions 
, the numbers of osteoclasts in myelomatous bones in our study did not increase during the experimental period. MM-related osteolysis results from an uncoupling of the processes of osteoclastic bone resorption and osteoblastic bone formation, which causes bone remodeling to shift toward bone destruction as activities of osteoclasts increase and of osteoblasts decrease 
. We speculate that, in these conditions, PTH contributes to restoring balance to the coupled bone-remodeling process in MM, which results in increasing the number of bone-building osteoblasts without altering the number of osteoclasts and, in some cases, even reducing the number of osteoclasts.
Indeed, GEP analysis demonstrates alterations in multiple signaling pathways that are critically involved in bone remodeling and in regulating the coupling of bone formation and bone resorption. To our knowledge, this is the first GEP analysis on human bones following treatment with PTH, and it may provide insight into additional mechanisms involved with this hormone's effects on bone. Interestingly, many of the differentially expressed genes are not expressed by the Hg myeloma cells and are thus considered microenvironment-associated genes.
The actions of PTH are mediated by a G protein-coupled receptor, PTH receptor 1 (PTHR1) 
. When PTH binds to the receptor, Gαs
-mediated activation of adenyl cyclase is stimulated, which then stimulates cAMP production and subsequent activation of protein kinase (PKA). Confirming activation of the cAMP/PKA pathway in myelomatous bones after PTH treatment, GEP analyses showed upregulation of cAMP-specific phosphodiesterases (e.g., PDE4D
), which are involved in regulating osteoblast production of PTH-induced cAMP 
. GEP also revealed upregulation of genes known to be stimulated by PTH treatment and/or associated with osteoblast differentiation, including RUNX2
(byglican), and various collagen types 
Although PTH is known to stimulate bone formation, the underlying mechanisms are not yet fully understood 
. Several studies have explored links between PTH and the Wnt signaling pathway 
, and our data confirm that Wnt signaling is involved in the anabolic activity of PTH in myelomatous bones. We identified increased expression of genes encoding several components of the canonical Wnt signaling pathway and target genes of the pathway, such as FZD1
, and CYR61
; we also noted downregulation of negative regulators of the Wnt pathway (i.e., CTBP1
, and DKK1
). Although reduced levels of two other Wnt signaling inhibitors, sclerostin (SOST
) and secreted frizzled related protein-2 (FRZB
), have been described in response to PTH 
, we did not detect changes in expression of those genes in our experimental setting.
The significant downregulation of DKK1
that resulted from PTH treatment emphasizes the critical role of this factor in myeloma-induced suppression of osteoblastogenesis 
. Because DKK1
is expressed by Hg myeloma cells and PTH treatment resulted in reduced growth of these cells in myelomatous bones, DKK1
downregulation could be a result of direct effects of PTH on osteoblasts, it could be an epiphenomenon of reduced tumor burden, or it could be a combination of both. Our data are consistent with recent studies demonstrating that PTH suppresses osteoblast production of DKK1 and that PTH can stimulate Wnt signaling and bone anabolism in the presence of DKK1 
. Interestingly, PTH reduced expression of LRP4
, which has been suggested to act as a sink and compete with Lrp5/6 for the binding of soluble Wnt antagonists (e.g., Wise, DKK1, and sclerostin) that are then not available to suppress the signal through the Lrp5/6 axis 
. These findings strongly indicate the important role of canonical Wnt signaling in the bone-anabolic effects of PTH.
Recent evidence shows that WNT5A activation of noncanonical Wnt signaling stimulates the osteogenic properties of human osteoblastic cells by homodimerization and activation of ROR2 
. We found that WNT5A
are significantly upregulated by PTH treatment, suggesting a role for noncanonical Wnt signaling in the anabolic effects of this hormone.
Recent studies suggest that bone-anabolic effects of PTH are amplified by bone marrow T lymphocytes secreting Wnt ligand Wnt10b 
. In our experimental system, Wnt10b
was expressed at very low levels in the human bones, and its expression was not affected by PTH treatment. Our data showed that PTH also increased BMD of the uninvolved femurs of SCID mice, which are deficient of T and B lymphocytes, suggesting that PTH effectively stimulates Wnt signaling in the absence of T lymphocytes.
Other Signaling Pathways
PTH resulted in upregulation of FOXC1
and downregulation of FOXO3
, and free-radical scavenging enzyme CAT
(catalase) in myelomatous bones. Initially it was suggested that oxidative stress antagonizes Wnt signaling in osteoblast precursors by diverting β-catenin from T cell factor-mediated transcription to FOXO-mediated transcription 
. FOXO transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis 
, which seems to be indispensable for bone homeostasis. Furthermore, decreasing oxidative stress levels normalizes bone formation and bone mass in mice lacking FoxO1 specifically in osteoblasts 
. These studies suggest that PTH reduced levels of oxidative stress in myelomatous bones, directly or indirectly, by reducing myeloma tumor burden and that regulation of bone remodeling by FOXO transcription factors depends on the physiological setting. Our data also suggest that other forkhead-related transcription factors, such as FOXC1
, may be involved in osteoblast apoptosis and bone homeostasis.
Although PTH has been shown to upregulate expression of ephrinB2 (EFNB2
) and to induce activation of ephrinB2/EphB4 forward signaling in murine osteoblasts 
, our GEP and qRT-PCR results showed insignificant increased expression of EFNB2
following PTH treatment. We previously demonstrated that EFNB2
are downregulated in osteoblast progenitors from patients with MM 
, partially explaining the lack of significant upregulation of EFNB2
in the current study.
PTH treatment affected expression of genes regulating bone remodeling through signaling pathways other than Wnt signaling. Upregulation of angiopoietin 1 and 2 and angiopoietin-like 2 is consistent with recent reports showing that angiopoietin 1 receptor, Tie2, is upregulated in differentiating osteoblasts and that angiopoietin 1 promotes bone formation 
. Upregulation of FGFR1
is consistent with previous studies and supports findings that FGF-2 signaling is critically important in the bone-anabolic effects of PTH 
. Upregulation of TGFB2
, and target genes of TGF (ID3
) strongly suggests a role for this signaling pathway in mediating the effects of PTH on bone formation and on coupling bone resorption to bone formation 
. Upregulation of PDGFA
by PTH treatment suggests involvement of this signaling pathway in the bone-anabolic effects of PTH and supports the notion of using PDGF as a therapeutic agent in treating bone loss associated with aging and fracture healing 
. Taken together, these results strongly suggest that PTH exerts bone anabolism in myelomatous bone through activation of multiple signaling pathways.
GEP analyses also revealed altered expression of a group of G protein-coupled receptors (e.g., GPR158) and phosphatase-related genes (e.g., PTPRD), but the functional association between PTH and these factors has yet not been elucidated.
Effects of PTH on Bone Marrow Microenvironment in MM
Despite the upregulation of RANKL by PTH, we did not observe differences in the numbers of osteoclasts or in expression levels of specific osteoclast markers (e.g., calcitonin receptor and cathepsin K). This phenomenon is not surprising. Lindsay et al 
described a significant increase in bone formation after one month of PTH treatment but no difference in the eroded perimeter or the osteoclast perimeter compared with controls. The apparent discrepancy could be the result of new bone that is both spatially and temporally unrelated to prior resorption or of bone formation extending to quiescent surfaces adjacent to the original resorption cavity 
. In experimental and clinical osteoporosis, increased bone formation without increased bone resorption often occurs in the initial stages of response to PTH, whereas catabolism occurs within the context of increased remodeling after approximately 6 months 
. Thus, the effects of long-term (>6 months) PTH treatment on MM bone disease should be carefully examined. Alternatively, PTH may be given in short cycles or in combination with antiresorptive agents to maximize the bone-forming effects and minimize potential proresorptive effects 
The GEP data provided important insights into molecular mechanisms that mediate reduced myeloma growth after PTH treatment. This hormone elicited marked increases in bone formation and increased numbers of mature osteoblasts, which express high levels of potential anti-tumor factors that include decorin, lumican, and CYR61. We recently demonstrated that mature osteoblasts negatively affect growth of myeloma cells 
and that this effect is partially mediated through production of decorin 
. Treatment with PTH had no effect on expression of myeloma cell growth factors, such as IL-6 and IGF-1, and PTH treatment did not stimulate myeloma growth in any of the experiments, even though we observed upregulation of VEGFB
. In addition to suggesting that PTH treatment may lessen oxidative stress in myelomatous bone, the GEP data indicate upregulation of anti-inflammatory factors, such as TNFAIP6
, and downregulation of inflammatory factor AIF1
. Recent study suggests that TNFAIP6
(also known as TSG-6) is an important anti-inflammatory factor that mediates improvement of myocardial infarction by systemic mesenchymal cell cytotherapy 
. Whereas CXCL14
suppresses tumor growth 
seems to promote tumor cell proliferation 
. These findings suggest that reduced inflammatory conditions contribute to PTH control of myeloma cell growth.
Current standard management for MM bone disease is limited to reducing tumor burden and treatment with bisphosphonates, and also often includes treatment with dexamethasone, a steroidal component that induces osteoporosis by reducing the life span of osteoblasts 
. Intriguingly, treatment with PTH has been shown to counteract the adverse effects of glucocorticoids on bone formation and strength 
. Bortezomib, the first proteasome inhibitor clinically approved for treating MM, stimulates bone formation in our experimental model 
and in patients with MM 
. Our current study suggests that combining treatments with PTH may abrogate dexamethasone-induced osteoporosis and act synergistically with proteasome inhibitors to stimulate bone formation and repair bone lesions in MM.
Collectively, the data presented here showed that PTH promotes bone formation in myelomatous bone by activating multiple distinct molecular pathways, of which Wnt signaling seems to play a major role. In vitro, PTH has no direct on effect on growth of myeloma cells, but in vivo PTH treatment indirectly attenuated MM progression by stimulating osteoblastogenesis and increasing osteoblast production of anti-myeloma factors, and by minimizing oxidative stress and inflammatory conditions in myelomatous bone. Our study supports the notion that MM and its associated bone disease are negatively impacted by alterations in the bone marrow microenvironment induced by osteoblast-activating agents.