Bone-resorbing cells in multiple myeloma: osteoclasts, myeloma cell polykaryons, or both?

Myeloma bone disease (MBD) leads to progressive destruction of the skeleton and is the most severe cause of morbidity in multiple myeloma. Its pathogenetic mechanisms are not fully understood, though the current evidence points to osteoclast (OC) hyperactivity coupled with defective osteoblast function unable to counteract bone resorption. OCs are generated in bone marrow by myeloid progenitors through increased levels of receptor activator of nuclear factor kappaB ligand and M-CSF, whose intracellular pathways propagate signals that activate sequential transcription factors, resulting in the production of major OC enzymes that drive specific functions such as acidification and degradation of the bone matrix. Osteolytic lesions, however, are not characterized by massive OC content, whereas malignant plasma cells, which are usually present in a high number, may occur as large multinucleated cells. The possibility that myeloma cells fuse and generate polykaryons in vivo is suggested by the in vitro formation of multinuclear cells that express tartrate-resistant acid phosphatase and produce pits and erosive lacunae on experimental osteologic substrates. Further, the detection in vivo of polykaryons with chromosome translocations typical of myeloma cells lends support to the view that myeloma polykaryons may act as functional OCs and participate in the skeletal destruction by resorbing bone.

Negative regulation of the osteoblast function in multiple myeloma through the repressor gene E4BP4 activated by malignant plasma cells.

PURPOSE: To explore the pathogenetic mechanisms that suppress the osteoblast function in multiple myeloma because osteogenesis results in defective new bone formation and repair. EXPERIMENTAL DESIGN: Microarray gene analysis revealed the overexpression of E4BP4, a transcriptional repressor gene, in normal osteoblasts cocultured with myeloma cells that were releasing the parathyroid hormone-related protein (PTHrP). Thus, the effect of E4BP4 was assessed in PTHrP-stimulated osteoblasts by measuring the RNA levels of both Runx2 and Osterix as major osteoblast transcriptional activators. Because E4BP4 is a negative regulator of the cyclooxygenase-2 (COX-2) pathway that drives the expression of both Runx2 and Osterix, these factors were investigated after prostaglandin E(2) treatment to overcome the COX-2 defect as well as in E4BP4-silenced osteoblasts. Finally, E4BP4, PTHrP, Osterix, and osteocalcin levels were measured in vivo in patients with bone disease together with the E4BP4 protein in bone biopsies. RESULTS: E4BP4 was specifically induced by PTHrP and inhibited both Runx2 and Osterix, whereas E4BP4-silenced osteoblasts expressed functional levels of both factors. The prostaglandin E(2) treatment of E4BP4-up-regulated osteoblasts promptly restored Runx2 and Osterix activities, suggesting that integrity of COX-2 pathway is essential for their transcription. Down-regulation of Osterix by E4BP4 was confirmed in vivo by its inverse levels in osteoblasts from myeloma patients with increased serum PTHrP, whose bone biopsies expressed the E4BP4 protein. CONCLUSIONS: Our data support the role of E4BP4 as osteoblast transcriptional repressor in inhibiting both Runx2 and Osterix in myeloma bone disease and correlate its effect with the increased PTHrP activity.

Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro.

Malignant plasma cells exert osteoclast-like activity in vitro. We investigated the function of the calcitonin (CT) receptor (R) on myeloma cells from patients and in myeloma cell lines. Primary myeloma cells expressed high CTR levels whereas the cell lines uniformly exposed the CTR-2 variant expressed by osteoclasts. Treatment of myeloma cell lines with CT modified the intracellular Ca(2+) and cAMP levels, suggesting the activation of both PKC and PKA pathways, and abrogated their bone resorptive property as erosive pits on osteologic substrates. Thus, the expression, sensitivity and function of CTR-2 in myeloma cells emphasize their osteoclast-like behavior in vitro.

[Mesenchymal stem cells and bone regeneration]. / Cellule staminali mesenchimali e medicina rigenerativa dell'osso.

Mesenchymal stem cells (MSC) are a cell population present not only in the bone marrow, but also in a number of adult and fetal tissues. Their multilineage differentiation in vitro emphasizes their potential usefulness in the field of the regenerative medicine. New techniques of molecular biology and genetic manipulation of MSC are under investigation for cell therapy of several bone diseases.

Myeloma bone disease: pathogenetic mechanisms and clinical assessment.

Bone disease in multiple myeloma (MM) leads to progressive devastation of the skeleton and is the most severe cause of morbidity. Its pathogenetic mechanisms are not fully defined, though the current evidence points to hyperactivation of osteoclasts (OC) in presence of a major defect of bone repairing in erosion sites due to osteoblast (OB) impairment. Bone resorption, however, is promoted by early OB, namely stromal cells that respond to chronic stimulation by myeloma cells by enhancing marrow levels of RANKL and other osteoclastogenic factors and thus accelerating the maturation of OC progenitors. In myeloma bone disease (MBD), OBs are systematically defeated by a number of inhibiting effects induced by the malignant clone within the marrow microenvironment. Thus, MBD primarily affects the OB lineage, particularly in overt MM, where serum markers of osteoblastogenesis, such as osteocalcin and osteoprotegerin, are extremely low in contrast with their slight increase in inactive MM. These markers, in association with others of bone turnover (RANKL, MIP-1alpha, type I collagen telopeptides such as NTX and CTX) may be used in the clinical assessment of MBD as well as to monitor the efficacy of bisphosphonate in delaying the progressive skeletal destruction.

In-vitro functional phenotypes of plasma cell lines from patients with multiple myeloma.

Seven plasma cell lines from patients with smoldering (group A) and overt myeloma (group B) were investigated for both phenotypic markers and in-vitro properties, including sensitivity to apoptosis, cytotoxicity, cell adhesion, chemotaxis and bone interaction. Cell lines from group A underwent apoptosis whereas those from group B were apparently resistant, promoted cytotoxicity in target cells and enhanced both adhesion and migratory functions upon appropriate activators. In addition, MCC-2, a group B cell line from a patient with severe osteolytic disease of the skeleton produced erosive lacunae on bone substrates, whereas this effect was almost absent with cell lines from group A. Concurrent deregulation of relative markers, in combination with peculiar properties including resistance to apoptosis and high cytotoxic potential, as well as adhesion, chemotaxis and bone pathophysiology interactions, may thus identify myeloma cells with aggressive phenotype driving these biological activities in vitro and perhaps in vivo.

PTHrP produced by myeloma plasma cells regulates their survival and pro-osteoclast activity for bone disease progression.

To promote their survival and progression in the skeleton, osteotropic malignancies of breast, lung, and prostate produce parathyroid hormone-related protein (PTHrP), which induces hypercalcemia. PTHrP serum elevations have also been described in multiple myeloma (MM), although their role is not well defined. When we investigated MM cells from patients and cell lines, we found that PTHrP and its receptor (PTH-R1) are highly expressed, and that PTHrP is secreted both as a full-length molecule and as small subunits. Among these subunits, the mid-region, including the nuclear localization sequence (NLS), exerted a proliferative effect because it was accumulated in nuclei of MM cells surviving in starvation conditions. This was confirmed by increased transcription of several genes enrolled in proliferation and apoptosis control. PTHrP was also found to stimulate PTH-R1 in MM cells. PTH-R1's selective activation by the full-length PTHrP molecule or the NH2 -terminal fragment resulted in a significant increase of intracellular Ca(2+) influx, cyclic adenosine monophosphate (cAMP) content, and expression of receptor activator of NF-κB ligand (RANKL) and monocyte chemoattractant protein-1 (MCP-1). Our data definitely clarify the role of PTHrP in MM. The PTHrP peptide is functionally secreted by malignant plasma cells and contributes to MM tumor biology and progression, both by intracrine maintenance of cell proliferation in stress conditions and by autocrine or paracrine stimulation of PTH-R1, which in turn reinforces the production of osteoclastogenic factors. © 2014 American Society for Bone and Mineral Research.

Bendamustine overcomes resistance to melphalan in myeloma cell lines by inducing cell death through mitotic catastrophe.

Melphalan has been a mainstay of multiple myeloma (MM) therapy for many years. However, following treatment with this alkylator, malignant plasma cells usually escape both apoptosis and cell cycle control, and acquire drug-resistance resulting in tumor progression. Bendamustine is being used in MM patients refractory to conventional DNA-damaging agents, although the mechanisms driving this lack of cross-resistance are still undefined. Here, we investigated the molecular pathway of bendamustine-induced cell death in melphalan-sensitive and melphalan-resistant MM cell lines. Bendamustine affected cell survival resulting in secondary necrosis, and prompted cell death primarily through caspase-2 activation. Also, bendamustine blocked the cell cycle in the G2/M phase and induced micronucleation, erratic chromosome spreading and mitotic spindle perturbations in melphalan-resistant MM cells. In these cells, both Aurora kinase A (AURKA) and Polo-like kinase-1 (PLK-1), key components of the spindle-assembly checkpoint, were down-regulated following incubation with bendamustine, whereas levels of Cyclin B1 increased as a consequence of the prolonged mitotic arrest induced by the drug. These findings indicate that, at least in vitro, bendamustine drives cell death by promoting mitotic catastrophe in melphalan-resistant MM cells. Hence, activation of this alternative pathway of cell death may be a novel approach to the treatment of apoptosis-resistant myelomas.

Constitutive down-regulation of Osterix in osteoblasts from myeloma patients: in vitro effect of Bortezomib and Lenalidomide.

Bortezomib and Lenalidomide have been shown to be effective in the control of multiple myeloma (MM) progression. We have investigated their role in the in vitro expression of Osterix by primary osteoblast cultures from MM patients and found that Osterix RNA was constitutively down-regulated in these cells. Treatment of osteoblasts with Bortezomib resulted in an increase of Osterix RNA and in enhanced activity of both BMP-2 and Runx2. Instead, Lenalidomide was unable to modify Osterix transcription. These findings provide additional evidence suggesting that, at least in vitro, Bortezomib promotes the osteoblast maturation whereas Lenalidomide is ineffective.

Umbilical cord mesenchymal stem cells: role of regulatory genes in their differentiation to osteoblasts.

Umbilical cord (UC) mesenchymal stem cells (MSCs) are being currently investigated as an alternative to bone marrow (BM) MSCs for bone repair and regeneration. Here, we describe the gene regulation of their differentiation to osteogenic, adipogenic, and chondrogenic precursors and demonstrate their tendency to differentiate toward the osteoblast lineage. Fibroblast-like cells from the Warthon's Jelly were cultured with dedicated media to obtain osteogenic-, adipogenic-, and chondrogenic-differentiated cells. After induction, a typical fibroblast-like shape with condensed fibers of F-actin was early noted in osteogenic-induced UC-MSCs, whereas those differentiating to adipocytes were flat with minor cytoskeleton relevance. Real-time PCR measured the transcription of master genes of the three lineages, thus revealing a remarkable up-regulation of Runx2 in osteogenic-induced cells with respect to both PPARg and SOX9 for adipogenic- and chondrogenic-differentiating UC-MSCs. However, TAZ, a coactivator of the nuclear transcription of Runx2 previously detected in BM-MSCs, was expressed in osteogenic- and, at lower magnitude, in adipogenic-induced cells, in keeping with its role in the reciprocal control of the differentiation between osteogenic- and adipogenic-induced cells. Its differential role in these cells was confirmed by its accumulation as protein product in the nuclei to activate Runx2 in osteogenic-differentiating UC-MSCs. These data emphasize the predominant expression by UC-MSCs of genes engaged in the osteogenic differentiation and their tendency to differentiate into osteoblasts, being similar in this respect to BM-MSCs. They may, thus, constitute a promising option for bone remodeling in regenerative medicine.

Functional expression of the calcitonin receptor by human T and B cells.

The calcitonin receptor (CTR) is a seven-transmembrane-domain G-protein-coupled receptor that regulates calcium metabolism and bone resorption by osteoclasts. Here we demonstrate that high levels are expressed by normal human T and B lymphocytes from tonsils and peripheral blood in relation to their activation status, as CTR(+) T cells are prone to produce IFN-gamma after TCR stimulation. The receptor is also highly expressed on B cells from chronic lymphocytic leukemia patients, thus suggesting a correlation between its expression, their proliferative extent as well as their memory, antigen-experienced phenotype. Moreover, we found that binding of the receptor with salmon calcitonin induces an increase of intracellular calcium(2+) in peripheral lymphocytes. This effect is involved in several lymphocyte immune functions, as cytosolic calcium(2+) levels regulate both cell proliferation and cytokine production. In our hands, the increase of calcium(2+) levels by CTR binding with sCT induced a dose-dependent cell proliferation. We therefore suppose that expression of this functional receptor may contribute to the modulation of cytoplasmic calcium(2+) levels needed to regulate T and B cell activation and perhaps other immune functions.