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Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple myeloma cells on osteoclastogenesis by upregulating the RANK expression.

ABSTRACT: Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor ?B ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.

SUBMITTER: Liu Z

PROVIDER: S-EPMC3858321 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple myeloma cells on osteoclastogenesis by upregulating the RANK expression.

Liu Zhiqiang Z Xu Jingda J Li Haiyan H Zheng Yuhuan Y He Jin J Liu Huan H Zhong Yuping Y Lu Yong Y Hong Bangxing B Zhang Mingjun M Lin Pei P Du Juan J Hou Jian J Qian Jianfei J Kwak Larry W LW Yi Qing Q Yang Jing J

PloS one 20131210 12

Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated ...[more]

PMID: 24340030

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Project description:INTRODUCTION: Recent studies indicate that bone marrow stromal cells (BMSCs) derived from patients with multiple myeloma (MM) differ from those of healthy donors in their expression of extracellular matrix compounds and in cytokine production. It is not known whether these abnormalities are primary or are acquired by BMSCs on contact with MM cells. MATERIALS AND METHODS: Interleukin (IL)-6, IL-11, IL-10, and tumor necrosis factor (TNF)-alpha production by CD166+ mesenchymal BMSCs and the CD38+/CD138+ RPMI8226 myeloma cell line cultivated in vitro in monocultures or co-cultivated under cell-to-cell contact or non-contact conditions in the presence of a tissue culture insert were measured. Intracellular cytokines were measured by flow cytometry analysis as the percentage of cytokine-producing cells or by mean fluorescence intensity as the level of cytokine expression in cells. Additionally, ELISA was used to measure IL-6, soluble IL-6 receptor (sIL-6R), IL-11, IL-10, TNF-alpha, B-cell-activating factor of the TNF family (BAFF), hepatocyte growth factor (HGF), and osteopontin (OPN) production in the supernatants of the cultures and co-cultures. RESULTS: A higher ability of the BMSCs of MM patients than in controls was detected to produce IL-6, IL-10, TNF-alpha, OPN, and especially HGF and BAFF in response to the RPMI8226 cells. Moreover, the BMSCs of the MM patients significantly enhanced the production of sIL-6R by the RPMI8226 cells. DISCUSSION: Cytokines over-expressed by BMSCs of MM patients can function as growth factors for myeloma cells (IL-6, IL-10, HGF), migration stimulatory factors for tumor plasma cells (TNF-alpha, HGF), adhesion stimulatory factors (HGF, BAFF and OPN), stimulators of osteoclastogenesis (IL-6, TNF-alpha), and angiogenic factors (TNF-alpha). The results of this experiment strongly suggest that the BMSCs from MM patients differed in spontaneous and myeloma cell-induced production of cytokines, especially of HGF and BAFF, and these abnormalities were both primary and acquired by the BMSCs on contact with the MM cells. This in turn suggests the presence of an undefined, autocrine stimulation pathway resulting in a prolonged production of cytokines even in long-term cultures in vitro and in vivo. These abnormalities might provide optimal conditions for the proliferation and differentiation of residual tumor cells or their precursors in the affected bone marrow.

Project description:Upon receptor activator of NF-?B ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560-565 (Motif 2) and PVQEQG604-609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK+/KI ) and wild-type (RANK+/+ ) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-?B, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-?B, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.

Dataset Information

Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple myeloma cells on osteoclastogenesis by upregulating the RANK expression.

Publications

Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple myeloma cells on osteoclastogenesis by upregulating the RANK expression.

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