Trimer Carboxyl Propeptide of Collagen I Produced by Mature Osteoblasts Is Chemotactic for Endothelial Cells

During the second phase of osteogenesis in vitro, rat osteoblasts secrete inducer(s) of chemotaxis and chemoinvasion of endothelial and tumor cells. We report here the characterization and purification from mature osteoblast conditioned medium of the agent chemotactic for endothelial cells. The chemoactive conditioned medium specifically induces directional migration of endothelial cells, not affecting the expression and activation of gelatinases, cell proliferation, and scattering. Directional migration induced in endothelial cells by conditioned medium from osteoblasts is inhibited by pertussis toxin, by blocking antibodies to integrins alpha(1), beta(1), and beta(3), and by antibodies to metalloproteinase 2 and 9. The biologically active purified protein has two sequences, coincident with the amino-terminal amino acids, respectively, of the alpha(1) and of the alpha(2) carboxyl propeptides of type I collagen, as physiologically produced by procollagen C proteinase. Antibodies to type I collagen and to the carboxyl terminus of alpha(1) or alpha(2) chains inhibit chemotaxis. The chemoattractant is the propeptide trimer carboxyl-terminal to type I collagen, and its activity is lost upon reduction. These data illustrate a previously unknown function for the carboxyl-terminal trimer, possibly relevant in promoting endothelial cell migration and vascularization of tissues producing collagen type I

PROTEASOME INHIBITORS MODULATE OSTEOCYTE DEATH AND AUTOPHAGY IN MULTIPLE MYELOMA.

Background: Cell death and autophagy are the main cellular processes involved in the regulation of bone remodeling by osteocytes. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis through the upregulation of osteoclast recruitment. Aims: Because proteasome inhibitors including Bortezomib (BOR) are known to be able to target osteoblasts in this study we have investigated the potential effect of these drugs on osteocytes and their cell death and autophagy. Methods: Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a transwell insert in the presence or the absence of BOR or MG262. Moreover the effect of proteasome inhibitors on dexamethasone (DEX)-induced MLO-Y4 death, obtained at high doses (10-5-10-6M), was checked in combination with PTH(1-34). To evaluate the presence of autophagy and apoptosis in osteocytes, we checked the expression of both autophagic marker LC3 and apoptotic marker APAF-1 by confocal microscopy in the co-colture system with MLO-Y4 and RPMI-8226. Finally we performed a retrospective histological evaluation on bone biopsies of a cohort of 31 newly diagnosis MM underwent to different treatments including BOR-based regimen. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. Results: The in vitro treatment with BOR or MG262 significantly blunted MLO-Y4 and HOB-01 cell death. Similarly, DEXinduced MLO-Y4 death was reduced by proteasome inhibitors. Interestingly, we found that both proteasome inhibitors potentiated the PTH (1-34) short-term effects on DEX-induced osteocyte death. Prevalence of autophagic cell death compared to apoptosis was observed in this system. In line with these data, we showed that neither the HMCLs nor treatment with DEX increase the apoptotic death and caspase 3 activation in both MLO-Y4 and HOB-01 cell lines. BOR treatment increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when the cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors blocked autophagic cell death in osteocytes. In the in vivo study we found a significant increase of the number of viable osteocytes in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Summary/Conclusion: Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy and potentiated the effect of PTH. Overall our in vitro and in vivo data support the use of BOR to improve bone integrity in MM patients

Myeloma-Induced Osteocyte Death Was Blunted By Proteasome Inhibitors Through The Modulation Of Autophagy

Osteocytes are critical in the maintenance of bone integrity regulating bone remodeling through the cell death and autophagy, a cellular process stress-induced to prolong cell survival but when induced excessively can cause cell death. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis. However the mechanisms involved in this process as well as the effect of the proteasome inhibitors able to stimulate bone formation are not known and have been investigated in this study. Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a traswell insert. The treatment for 12-24 hours with (BOR) (2nM) and MG262 (10nM) significantly blunted MLO-Y4 and HOB-01 cell death. In addition, dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at high doses (10-5-10-6 M), was reduced by the treatment with proteasome inhibitors. Interestingly, we found that PTH short-term treatment potentiated the in vitro effects of proteasome inhibitors on DEX-induced osteocyte death. To evaluate the presence of autophagy in osteocytes, we checked the expression of the autophagic marker LC3 both by confocal microscopy and western blot analysis in the co-colture system with MLO-Y4 and RPMI-8226. Prevalence of autophagic cell death and in a lesser extent apoptosis was observed in this system. BOR increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors BOR and MG262 blocked autophagic cell death in osteocytes. To translate our in vitro evidence in a clinical perspective, thereafter we performed a histological evaluation on bone biopsies of a cohort of 37 newly diagnosis MM patients 31 of them with symptomatic MM and 6 with smoldering MM (SMM). The 55% of patients with MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment or observation. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). A reduction of both osteocyte apoptosis and autophagy was demonstrated by TUNEL assays and confocal microscopy. On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy supporting their use to improve bone integrity in MM patients

Proteasome Inhibitors Block Myeloma-Induced Osteocyte Death in Vitro and in Vivo in Multiple Myeloma Patients

Multiple myeloma (MM) is characterized by a severe unbalanced and uncoupling bone remodeling leading to osteolysis. We have recently shown that osteocytes are involved in MM-induced osteolysis through an increased cell death. Accordingly MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions. Proteasome inhibitors currently used in the treatment of MM are able to stimulate osteoblast formation but their potential effects on osteocyte death are not known and have been investigated in this study both in vitro and in vivo. Osteocytic MLO-Y4 cells or human pre-osteocytic HOB-01 cells were co-cultured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) JJN3 or RPMI-8226 placed in a transwell insert. A significantly reduction of ostecyte viability was observed (median percent reduction of MLO-Y4 viability: -16% and -30%, respectively). The treatment for 12–24 hours with Bortezomib (BOR) (2nM) or other proteasome inhibitors such as MG262 (10nM) or MG132 (100nM) significantly blunted MLO-Y4 and HOB-01 cell death. Similarly, Dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at pharmacological doses (10–4–10–5 M), was significantly reduced by the treatment with proteasome inhibitors. To translate our in vitro data into a clinical perspective we performed a retrospective histological evaluation on bone biopsies of a cohort of 40 newly diagnosis MM patients (24 male and 16 female, median age: 68 years) 34 of them with symptomatic MM and 6 with smoldering MM (SMM). The 58% of patients with symptomatic MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained in both symptomatic MM and SMM at diagnosis and after an average time of 12 months of treatment or observation, respectively. The 68% of patients with symptomatic MM were treated with a BOR-based regimen while 42% do not. Moreover the 58% of MM patients received DEX and the 59% Thalidomide (TAL). Zoledronic acid (ZOL) was infused monthly in the 60% of MM patients. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections, corresponds to the total number of osteocyte lacunae in the bone biopsies. The number of viable osteocytes and the number of degenerated or apoptotic osteocytes and empty lacunae have been evaluated. In patients with SMM no significant change was observed in the number of viable osteocytes in the two histological evaluations carried out (median percent change: +1.2, p=0.68, NS). In symptomatic MM patients the mean percent change of the osteocyte viability was not correlated with the response rate to treatment (R2 0.01, p=NS). A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase of osteocyte viability: +6% vs. +1.30%, Mann-Whitney test: p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability that was statistical significant in comparison with that observed either in patients treated without BOR (+11.6% vs. +1.3%, p=0.0019) or in those treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). On the contrary, no significant difference was observed in patients treated with TAL than in those treated without TAL (p= 0.7, NS) as well as patients treated with ZOL compared to those untreated showed no significant difference in the number of viable osteocytes (p=0.18, NS). To confirm the role of the different drug treatment on the osteocyte viability we perform a multiple regression non-parametric analysis showing that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas ZOL and TAL have not (p>0.2,NS) and it counterbalanced the negative effect of DEX treatment (p=0.035). In conclusion our in vitro and in vivo data suggest the proteasome inhibitors block osteocyte death induced by MM cells could have a positive impact on bone integrity in MM patients

The anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells.

International audienceThe anti-tumoral effect of lenalidomide is increased in vivo by hypoxia-inducible factor (HIF)-1α inhibition in myeloma cells We investigated the effect of stable suppression of hypoxia inducible factor (HIF)-1α in myeloma cells on sensitivity to lenalidomide (LEN) in vivo. We found that the in vivo anti-tumoral effect of LEN is enhanced by HIF-1α suppression in myeloma cells. It has been reported that HIF-1α is over-expressed by myeloma cells 1-4 and that HIF-1α suppression significantly blocks myeloma-induced angiogenesis, and reduces both tumoral burden and bone destruction in vivo in multiple myeloma (MM) mouse models. 3 The potential effects of HIF-1α modulation on drug sensitivity in MM cells are not known and are currently under investigation. The immunomodulatory drugs (IMiDs®), including LEN, are a class of drugs derived from thalidomide 4 able to exert anti-myeloma effects by the selective Cereblon-dependent destruction of IKZF proteins, 5,6 either through a direct action on MM cell proliferation and survival, 7 or through indirect immunomodulatory and anti-angiogenic effects. 7 Previous data indicated that HIF-1α inhibition did not increase the anti-proliferative in vitro effect of bortezomib on MM cells; 2,3 this drug induces a strong downregula-tion of HIF-1α in MM cells. 2 We recently reported that HIF-1α knockdown in the human myeloma cell line (HMCL) JJN3 potentiated the in vitro effect of LEN treatment (48-72 h) on cell proliferation through a significant upregulation of p27 without changing cell viability. 3 It has been consistently reported that LEN only slightly down-regulated HIF-1α expression in MM cells. 2 Such evidence has provided the rationale to investigate the effect of stable suppression of HIF-1α in myeloma cells on LEN sensitivity in vivo. Therefore, in the present study, we first inhibited HIF-1α in three HMCLs (JJN3, OPM2 and H929) using an anti-HIF1α lentiviral shRNA pool, as previously described. 3 H929 showed a very high mortality rate after anti-HIF1α lentiviral infection (data not shown) and were not used for the subsequent in vivo experiments. We assessed the effect of LEN in combination with HIF-1α inhibition in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) subcutaneous in vivo mouse model. 3 Different groups of animals (5 animals in each group) of two sets of independent experiments were injected with JJN3 pLKO.1 (empty vector) or JJN3 anti-HIF1α. When tumors became palpable (approx. 7-10 days after injection) mice were treated with LEN 5 mg/kg (Selleckchem, Houston, TX, USA) or vehicle (DMSO) using the intraperitoneal route. The same mouse model, grouping strategy and treatment conditions were used for OPM2 pLKO.1 or OPM2 anti-HIF1α. After three weeks, we evaluated tumor volume and weight, as previously described, 3 and immunohisto-chemistry was used to evaluate the microvascular density (MVD) and checked by CD34 immunostaining (Santa Cruz, Dallas, TX, USA). 3 In addition, in the first set of mice experiments, the expression of p27 (Abcam, Cambridge, UK) was evaluated by immunohistochem-istry. The expression of S-phase kinase-associated protein 2 (SKP2), a p27 inhibitor, expression of the HIF-1α target key mediator of glycolysis and tumoral growth, Hexokinase II (HK2), and levels of pERK 1/2, and total Caspase-3 (Casp-3) were evaluated in the ex vivo plasma-cytoma lysates by western blot using the following anti-bodies: SKP2, Casp-3 (Santa Cruz, Dallas, TX, USA), HK2, pERK 1/2, (Cell Signaling, Danvers, MA, USA). b-actin was used as internal control (Millipore