MIP-1alpha utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells

OBJECTIVES: Macrophage inflammatory protein-1alpha (MIP-1alpha), an osteoclast (OCL) stimulatory factor produced by primary multiple myeloma (MM) cells, increases bone destruction and tumor burden in murine models of MM. Several chemokine receptors (CCR1, CCR5, and CCR9) mediate the effects of MIP-1alpha. In this study, we determined which of these mediates the effects of MIP-1alpha on human OCL formation and myeloma cells. METHODS: We employed RT-PCR analysis, neutralizing antibodies to CCR1 and CCR5 as well as a CCR1-specific antagonist and OCL formation assays to identify the MIP-1alpha receptors involved in MIP-1alpha's effects on myeloma cells and OCL formation. RESULTS: RT-PCR analysis demonstrated that both CCR1 and CCR5 were expressed by highly purified human OCL precursors, myeloma cell lines, and purified marrow plasma cells from MM patients. Neutralizing antibodies to CCR1 or CCR5 inhibited MIP-1alpha-induced OCL formation. Furthermore, monocyte chemotactic protein-3 (MCP-3), which binds CCR1 but not CCR5 and the CCR1-specific antagonist, BX471, markedly inhibited OCL formation stimulated with MIP-1alpha. Anti-CCR1, anti-CCR5, or BX471 also inhibited the upregulation of beta1 integrin mRNA in myeloma cells induced by MIP-1alpha, as well as the adherence of myeloma cells to stromal cells and IL-6 production by stromal cells in response to myeloma cells. CONCLUSION: These data demonstrate that MIP-1alpha utilizes either CCR1 or CCR5 for its effects on OCL formation and myeloma cells, and that blocking either CCR1 or CCR5 inhibits OCL formation and myeloma cell adhesion to stromal cells.     

Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation

BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box-binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.     

Arginine increases growth hormone gene expression in rat pituitary and GH3 cells

The effect of arginine (Arg) and Ornitargin (OT) [a compound containing the aminoacids Arg, citrulline (Cit) and ornithine (Orn)] administration upon growth hormone (GH) gene expression was studied both in vivo and in vitro (hemipituitaries and GH3 cells) by Northern blot analysis. For in vivo studies, adult male Wistar rats were anesthetized, subjected to i.v. infusion of 200 microl of 150 mM NaCl (control group), Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) at a rate of 20 microl/min, and killed 50 min thereafter. For the in vitro studies, hemipituitaries or GH3 cells were incubated in 1 ml of appropriate medium containing Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) for 60 min. The pituitaries of the in vivo and in vitro studies and GH3 cells were subsequently processed for RNA extraction. Total RNA was subjected to electrophoresis in agarose (1%)/formaldehyde gel, transferred to a nylon membrane and subjected to hybridization with a rat GH (32)P-cDNA, and (32)P-18S rRNA probe to correct for the variability in RNA loading. After autoradiography of the membrane, the abundance of GH mRNA and 18S rRNA bands was quantified by densitometry. The in vivo study demonstrated that Arg and OT infusion induced a 2.3-fold increase in GH mRNA expression, which could result from the Arg-mediated inhibition of somatostatin release. In addition, in vitro Arg, but not OT, induced GH gene expression in hemipituitaries and GH3 cells, indicating that the aminoacid can act per se at the pituitary somatotrope level. In conclusion, our data show for the first time that arginine stimulates GH gene expression in parallel to its recognized GH-releasing activity.     

PRL-2 increases Epo and IL-3 responses in hematopoietic cells

Dual specificity protein tyrosine phosphatase PRL-2 is overexpressed in pediatric acute myeloid leukemia (AML) and is located at human chromosome 1p35, a region often rearranged or amplified in malignant lymphoma and B-cell chronic lymphocytic leukemia (B-CLL). Little is known of the significance of PRL-2 expression in hematopoietic malignancies. Herein we demonstrated that ectopic expression of PRL-2 in murine pre-B-cell line Baf3ER and mouse bone marrow cells induced key features associated with malignant progression and metastasis. PRL-2-transfected Baf3ER cells had augmented growth responses to hematopoietic growth factors Epo or IL-3 with shortened cell cycle, reduced requirement (5×) for Epo in cell survival, increased cell migration (3×), reduced cell adhesion (5×), and conversion to an immature cell morphology in association with increased expression (3×) of stem cell marker Bmi-1. When transduced into mouse bone marrow cells, PRL-2 increased Epo-induced colony formation (4×) and gave rise to larger colonies. These observations provide evidences implicating PRL-2 as a pathogenic molecule in hematopoietic malignancies and suggest its potential as a novel therapeutic target.     

Pleiotrophin is highly expressed by myeloma cells and promotes myeloma tumor growth

Pleiotrophin (PTN) is an important developmental cytokine that is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is largely restricted to the brain. High PTN serum levels are associated with a variety of solid tumors. We recently showed that patients with multiple myeloma (MM) also have elevated serum levels of this protein and the amount of PTN correlated with the patients' disease status and response to treatment. In this study, we demonstrate that MM cell lines and the malignant cells from MM patients' bone marrow produced PTN and secreted PTN protein into the supernatants during short-term culture. Moreover, Ptn gene expression correlated with the patients' disease status. Inhibition of PTN with a polyclonal anti-PTN antibody reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro. Importantly, this antibody also markedly suppressed the growth of MM in vivo using a severe combined immunodeficiency (SCID)-hu murine model. This represents the first study showing the importance of PTN in the growth of any hematological disorder. Because the expression of this protein is very limited in normal adult tissues, PTN may represent a new target for the treatment of MM.     

Osteoblastic cells are involved in osteoclast formation

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.     

The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated

The one or more molecular mechanisms that determine the obligatory sequence of resorption followed by formation during bone remodeling is unclear. RANK ligand (RANK-L) is an essential requirement for osteoclastogenesis, and its activity is neutralized by binding to the soluble decoy receptor, osteoprotegerin (OPG). Because both molecules are produced by osteoblast lineage cells, we studied their developmental regulation in a conditionally immortalized human marrow stromal (hMS[2-15]) cell line. These cells can simulate the complete developmental sequence from undifferentiated precursor(s) to cells with the complete osteoblast phenotype that are capable of forming mineralized nodules. During osteoblast differentiation, RANK-L messenger RNA levels decreased by 5-fold, whereas OPG messenger RNA levels increased by 7-fold, resulting in a 35-fold change in the RANK-L/OPG ratio. OPG protein also increased by 6-fold. Mouse bone marrow cells generated osteoclast-like cells in coculture with undifferentiated hMS(2-15) cells, but did not when cocultured with hMS(2-15) cells in varying stages of differentiation, unless an excess of RANK-L was added. Thus, undifferentiated marrow stromal cells with a high RANK-L/OPG ratio can initiate and support osteoclastogenesis, but after differentiation to the mature osteoblast phenotype, they cannot. We speculate that the developmental regulation of OPG and RANK-L production by stromal/osteoblast cells contributes to the coordinated sequence of osteoclast and osteoblast differentiation during the bone remodeling cycle.     

gamma-Interferon in multiple myeloma: inhibition of interleukin-6 (IL- 6)-dependent myeloma cell growth and downregulation of IL-6-receptor expression in vitro

In multiple myeloma, malignant plasma cells from most patients with active disease proliferate spontaneously when cultured for 5 days in vitro. This spontaneous proliferation is related to the endogenous production of interleukin-6 (IL-6), the major myeloma-cell growth factor. A 50% inhibitory dose (100 U/mL) of human recombinant gamma- interferon (hr gamma-IFN) blocked the proliferation of myeloma cells almost completely in all 19 patients analyzed. This inhibition was not caused by suppression of endogenous IL-6 production and was also observed in the presence of an excess of hrIL-6. hr gamma-IFN was also completely inhibitory in four human myeloma cell lines (HMCL) whose growth is totally dependent on the addition of exogenous hrIL-6. This inhibition was associated with a 47% to 73% decrease in membrane IL-6- binding gp80 protein as well as with a 90% decrease in the amount of gp80 mRNA in HMCL. These results are in line with recent reports indicating that gamma-IFN inhibited several IL-6-dependent biologic processes. They suggest a need to reconsider why previous preliminary clinical trials failed to demonstrate a beneficial effect of gamma-IFN in multiple myeloma.     

IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor gamma 1

IL-4 is a pleiotropic immune cytokine secreted by activated T(H)2 cells that inhibits bone resorption both in vitro and in vivo. The cellular targets of IL-4 action as well as its intracellular mechanism of action remain to be determined. We show here that IL-4 inhibits receptor activator of NF-kappaB ligand-induced osteoclast differentiation through an action on osteoclast precursors that is independent of stromal cells. Interestingly, this inhibitory effect can be mimicked by both natural as well as synthetic peroxisome proliferator-activated receptor gamma1 (PPARgamma1) ligands and can be blocked by the irreversible PPARgamma antagonist GW 9662. These findings suggest that the actions of IL-4 on osteoclast differentiation are mediated by PPARgamma1, an interpretation strengthened by the observation that IL-4 can activate a PPARgamma1-sensitive luciferase reporter gene in RAW264.7 cells. We also show that inhibitors of enzymes such as 12/15-lipoxygenase and the cyclooxygenases that produce known PPARgamma1 ligands do not abrogate the IL-4 effect. These findings, together with the observation that bone marrow cells from 12/15-lipoxygenase-deficient mice retain sensitivity to IL-4, suggest that the cytokine may induce novel PPARgamma1 ligands. Our results reveal that PPARgamma1 plays an important role in the suppression of osteoclast formation by IL-4 and may explain the beneficial effects of the thiazolidinedione class of PPARgamma1 ligands on bone loss in diabetic patients.     

Mcl-1 and Bcl-xL are co-regulated by IL-6 in human myeloma cells

Multiple myeloma (MM) is a slowly proliferative malignancy in which malignant plasma cells accumulate within the bone marrow. The expression of several anti-apoptotic proteins was evaluated by immunoblotting in human myeloma cell lines and in highly purified native myeloma cells. Expression of Bcl-xL, Mcl-1 and Bcl-2 was found in most of the samples; expression of Bcl-xL and Mcl-1 seemed to be related on myeloma cells. In a system of apoptosis by growth factor deprivation on myeloma cells, we showed that the effect of Bcl-2 seemed minimal whereas Mcl-1 and Bcl-xL were tightly regulated by interleukin (IL)-6. These findings underline the important role of Mcl-1 and Bcl-xL instead of Bcl-2 in IL-6-induced survival of myeloma cells.     

Up-regulation of IL-6-receptors by IL-3 on a plasma cell leukaemia cell line which proliferates dependently on both IL-3 and IL-6

Summary. In this report we show that IL-3 up-regulates the expression of IL-6-receptors on a plasma cell leukaemia cell line which proliferates in response to both IL-3 and IL-6. Quantitative binding studies and Scatchard analysis revealed that HSM-2.3 has a single class of high-affinity IL-6 binding sites (200/cell, dissociation constant at the equilibrium 1.43 × 10⁻¹¹ m). After stimulation with IL-3, HSM-2.3 has 1200 binding sites. IL-6 receptor mRNA expression was detected in HSM-2.3 only after stimulation with IL-3.     

Up-regulation of vinculin expression in 3T3 preadipose cells by growth hormone

In an effort to define biochemical events relevant to the adipogenic action of GH, the effect of GH on expression of the cytoskeletal element vinculin was studied in 3T3-F442A preadipose cells. Results from Western blotting indicated that in serum-free medium 2 nM met-hGH induced an approximately 100% increase in vinculin expression relative to that in cells maintained in serum-free medium alone. GH-elicited alterations in vinculin expression were dose dependent. GH treatment elevated levels of tubulin to a lesser extent, whereas actin expression was unaffected by GH. Immunoprecipitation experiments revealed that GH treatment promoted a 200% increase in vinculin synthesis on day 4 relative to that in control cells. GH had no effect on phosphorylation of vinculin in 3T3-F442A cells. Based on Northern blotting, we noted that GH induced approximately a 200% increase in levels of vinculin mRNA on day 4. GH responsiveness as well as levels of vinculin were similar in 3T3-GI-16 (a highly adipogenic subclone of the 3T3-F442A cell) and 3T3-F442A cells. The GH-dependent increase in vinculin protein expression was not observed in nonadipogenic 3T3-C2 cells, suggesting that this effect of GH was related to the program of differentiation. Interestingly, levels of vinculin in nontreated 3T3-C2 cells were approximately 10-fold lower than levels in 3T3-F442A cells. GH-mediated alterations in vinculin expression in 3T3-F442A cells were abolished by treatment with fetal bovine serum, a potent mitogen. Our data indicate that increased expression of vinculin is a component of the GH-induced portion of the adipose differentiation program.     

IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation

Thymic-dependent differentiation of bone marrow (BM)-derived progenitors and thymic-independent antigen-driven peripheral expansion of mature T cells represent the 2 primary pathways for T-cell regeneration. These pathways are interregulated such that peripheral T-cell expansion is increased in thymectomized versus thymus-bearing hosts after bone marrow transplantation (BMT). This study shows that this interregulation is due to competition between progeny of these 2 pathways because depletion of thymic progeny leads to increased peripheral expansion in thymus-bearing hosts. To test the hypothesis that competition for growth factors modulates the magnitude of antigen-driven peripheral expansion during immune reconstitution in vivo, a variety of T-cell active cytokines were administered after BMT. Of the cytokines (interleukins) tested (IL-3, IL-12, IL-6, IL-2, and IL-7), IL-2 modestly increased peripheral expansion in the face of increasing numbers of thymic emigrants, whereas IL-7 potently accomplished this. This report also demonstrates that the beneficial effect of IL-7 on immune reconstitution is related to both increases in thymopoiesis as well as a direct increase in the magnitude of antigen-driven peripheral expansion. Therefore, the administration of exogenous IL-7, and to a lesser extent IL-2, abrogates the down-regulation in antigen-driven peripheral expansion that occurs in thymus-bearing hosts after BMT. These results suggest that one mechanism by which T-cell-depleted hosts may support antigen-driven T-cell expansion in vivo is via an increased availability of T-cell-active cytokines to support clonal expansion.     

Iron increases the susceptibility of multiple myeloma cells to bortezomib

Multiple myeloma is a malignant still incurable plasma cell disorder. Pharmacological treatment based on proteasome inhibition has improved patient outcome; however, bortezomib-resistance remains a major clinical problem. Inhibition of proteasome functionality affects cellular iron homeostasis and iron is a potent inducer of reactive oxygen species and cell death, unless safely stored in ferritin. We explored the potential role of iron in bortezomib-resistance. We analyzed iron proteins, oxidative status and cell viability in 7 multiple myeloma cell lines and in plasma cells from 5 patients. Cells were treated with increasing bortezomib concentrations with or without iron supplementation. We reduced ferritin levels by both shRNA technology and by drug-induced iron starvation. Multiple myeloma cell lines are characterized by distinct ferritin levels, which directly correlate with bortezomib resistance. We observed that iron supplementation upon bortezomib promotes protein oxidation and cell death, and that iron toxicity inversely correlates with basal ferritin levels. Bortezomib prevents ferritin upregulation in response to iron, thus limiting the ability to buffer reactive oxygen species. Consequently, reduction of basal ferritin levels increases both bortezomib sensitivity and iron toxicity. In patients’ cells, we confirmed that bortezomib prevents ferritin increase, that iron supplementation upon bortezomib increases cell death and that ferritin reduction overcomes bortezomib resistance. Bortezomib affects iron homeostasis, sensitizing cells to oxidative damage. Modulation of iron status is a strategy worth exploring to improve the efficacy of proteasome inhibition therapies.     

The growth fraction of human myeloma cells

Greater reductions of tumor load in patients with multiple myeloma may result from therapeutic strategies that are based on a better knowledge of growth kinetics. We have previously shown that the labeling index of myeloma cells remains unchanged when tumor mass is reduced and that the cells of relapsing patients have differnt biologic properties than the cells present before melphalan-prednisone therapy. This study investigated the growth fraction (GF) of myeloma cells at various disease stages using continuous i.v. infusions of tritiated thymidine. We studied 17 patients on 22 occasions (4 untreated, 2 unresponsive, 6 in remission, and 10 in relapse). All untreated an unresponsive patients and 5 of 6 patients in remission had a GF of less than 4%. GF was defined in these studies as the maximum percentage of labeled plasma cells exposed continuously to tritiated thymidine. Relapsing patients, with the most rapid tumor doubling times, had GF ranging from 14% to 83%. The plasma cell transit time through the proliferative compartment for all of the relapsing patients ranged from 6.6 to 11.9 days and the calculated intrinsic cell loss ranged from 50% to 86%. These findings support our model for the growth kinetics of multiple myeloma that assumes that the entire tumor mass issues from a small proportion of proliferating cells and that the growth kinetics of myeloma cells in relapsing patterns differ from those in untreated and unresponsive patients. Therapeutic trials with cycle-active agents need further investigation in selected relapsing patients who are likely to have a high growth fraction.