HOBIT Cells Research Articles

Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions.Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions.We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression.Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested.Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. DisclosuresAversa:Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

Effects of Antiretroviral Molecules on Survival and Gene Expression of An Osteoblast-like Cell Line.

The advent of combined antiretroviral therapy effectively undermined the evolution of HIV disease. Nevertheless, clinical observations indicated a clear association between therapy and the impairment of bone mineral density. We selected some antiretroviral compounds used in clinical practice, to study their impact on bone health and their possible implication in the onset of bone disease. Scalar concentrations of several antiretroviral drugs (used in single and in combination) were tested on an osteoblast-like cell line, HOBIT cells, to analyse cell survival and gene expression of selected bone markers. None of the tested concentrations of Tenofovir, Emtricitabine, Nevirapine, Maraviroc or Raltegravir induced any significant apoptosis activation at our experimental conditions. Only some protease inhibitors and Efavirenz, at high concentration, determined a significant activation of programmed cell death. In parallel experiments, protease inhibitors used in combination with Tenofovir and Emtricitabine, increased apoptosis. Furthermore, we performed a study of mRNA expression of specific genes involved in osteoblast biology and in bone synthesis and observed that some protease inhibitors induced a selective decrease of some osteogenic markers. All the protease inhibitors included in this study trigger apoptosis at the highest concentration analysed, suggesting great caution in HIV-patients co-infected with HBV or HCV, where elevated plasma concentrations of drugs could be reached as a consequence of liver failure. Lastly, an increased apoptosis rate and an impairment of osteogenic markers were recorded only in the presence of Nelfinavir, suggesting a role of protease inhibitors in the alteration of osteoblast biology.

Cells of the Osteoblast Lineage Confer Myeloma Cell Resistance to Established and Investigational Therapeutic Agents

AbstractAbstract 3995 Introduction:Multiple myeloma (MM) cells suppress osteoblast (OB) maturation and function and induce osteoclast-mediated bone resorption. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present in the MM bone microenvironment and their impact on MM cell response to treatment has not been fully characterized. We therefore studied human immortalized cells of the osteoblast lineage as surrogate models for in vitro studies on the effects of pre-osteoblasts on MM cell proliferation, survival and drug resistance. Methods:Immortalized hFOB 1.19 (here referred to as hFOB) and HOBIT cells were utilized in our studies, because, in the absence of Dexamethasone or vitamin D, they express markers consistent with pre-osteoblast stage of differentiation (high type I collagen levels; and low alkaline phosphatase, osteocalcin and osteopontin expression). Monolayer cultures of hFOB cells were performed to determine drug concentrations that did not exhibit cytotoxic effects on these pre-osteoblasts. Next, co-cultures of hFOB and HOBIT cells were performed using a panel of tumor cell lines from MM (n=13), breast (n=1) and lung (n=1) cancer. Compartment-specific bioluminescence imaging (CS-BLI) assays were undertaken to quantify the proliferative responses of the tumor cells in the presence of pre-osteoblasts, and if any changes are observed in tumor cell responses to established or novel therapeutic agents. Assays with Transwell insert chambers were conducted to allow for culture of tumor cells proximate to pre-osteoblasts, but preclude their direct cell-to-cell contact, in order to determine whether paracrine factors released by pre-osteoblasts are sufficient to recapitulate the effect(s) of their co-culture with tumor cells. Finally, cytokine or cytokine receptor neutralization studies with monoclonal antibodies or selective kinase inhibitors were performed to investigate the functional contribution of IL-6 or IGF1R-mediated signalling in tumor-pre-osteoblast interactions. Results:Pharmacologically relevant doses of conventional and novel therapies did not exhibit substantial cytotoxic effects on hFOB cells. Within 48hrs of co-culture, pre-osteoblasts stimulated proliferation of several tumor cell lines, including MM.1S, MM.1R, RPMI8226 and Dox40 in co-culture with hFOB; and NCI-H929, OPM2, H23 and Dox40 with HOBIT co-culture. hFOB cells decreased the sensitivity of 8 MM cell lines to at least one established (doxorubicin, dexamethasone, lenalidomide) or novel (vorinostat, pomalidomide) therapy tested. These observations were particularly pronounced in MM.1S and KMS34 cell lines. Subsequent Transwell-based co-culture assays showed that lack of direct cell-to-cell contact significantly decreases or even completely abrogates the resistance observed in respect to doxorubicin, lenalidomide or vorinostat in mono-cultures of tumor cells with pre-osteoblasts. Finally, IL-6 neutralization with monoclonal antibody did not overcome the proliferative effect of hFOB on MM cells. The anti-MM effect of IGF1R kinase inhibitors was also suppressed by co-culture with hFOB cells. These 2 latter results taken together indicate that the protection provided by hFOB cells is independent of IL-6 and IGF-1R-signaling in tumor or pre-osteoblasts. Conclusion:Our results suggest that cells of the osteoblast lineage may play a role as promoters of MM cell survival and resistance to diverse established and investigational therapeutics. Ongoing mechanistic and functional validation studies aim to delineate new therapeutic strategies to overcome pre-osteoblast-mediated drug resistance in MM and, potentially, other neoplasias. Disclosures:Richardson:Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity’s Board of Directors or advisory committees; Oncopep: Membership on an entity’s Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor Other.

HIV-1 Induces Apoptosis in Primary Osteoblasts and HOBIT Cells through TNF-alfa Activation

HIV-1 Induces Apoptosis in Primary Osteoblasts and HOBIT Cells through TNF-alfa Activation

HIV‐1 triggers apoptosis in primary osteoblasts and HOBIT cells through TNFα activation

Several HIV-1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV-1-mediated direct and/or indirect effects on osteoblasts/osteoclasts cross-talk regulation. This study investigated the effects of HIV-1(IIIb) and HIV-1(ADA) strains on osteoblasts using the osteoblast-derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV-1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV-1 DNA and supernatant HIV-1 RNA were not detected by real time PCR or b-DNA assays respectively. Under the experimental conditions, even heat-inactivated HIV-1 or cross-linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV-1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up-regulation of TNFalpha, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti-TNFalpha polyclonal antibody tackled effectively HIV-1 related induction of cell apoptosis. As a whole, these results indicate that HIV-1 may impair bone mass structure homeostasis by TNFalpha regulated osteoblast apoptosis.

Sparc localizes to the blebs of hobit cells and human primary osteoblasts

Secreted protein acidic and rich in cystein (SPARC) is a secreted glycoprotein involved in several biological processes such as tissue remodeling, embryonic development, cell/extracellular matrix interactions, and cell migration. In particular, SPARC affects bone remodeling through the regulation of both differentiation/survival of osteoblasts and bone extracellular matrix synthesis/turnover. Here, we investigated SPARC subcellular localization in the human osteoblastic HOBIT cell line by immunocytochemistry and western blot analysis. We show that, under normal exponential cell growth conditions, SPARC localized both to cell nucleus and to cytoplasm, with no co-localization on actin stress fibers. However, in colchicine-treated HOBIT cells and human primary osteoblasts undergoing blebs formation, SPARC showed a different cellular distribution, with an additional marked compartmentalization inside the blebs, where it co-localized with globular actin and actin-binding proteins such as alpha-actinin, cortactin, and vinculin. Moreover, we demonstrate by an in vitro assay that the addition of SPARC to actin and alpha-actinin inhibited the formation of cross-linked actin filaments and disrupted newly formed filaments, most likely due to a direct interaction between SPARC and alpha-actinin, as indicated by immunoprecipitation assay. The specific silencing of SPARC RNA expression markedly decreased the ability of colchicine-treated HOBIT cells to undergo blebbing, suggesting a direct role for SPARC in cell morphology dynamics during cytoskeletal reorganization.

H 2O 2 modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H 2O 2 and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H 2O 2 induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H 2O 2 doses. The H 2O 2-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H 2O 2 failed to evoke calcium signals and millimolar H 2O 2 induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H 2O 2 up to 1 mM did not increase the cytosolic calcium concentration. In ROS/P2Y 2 cells, stably expressing the P2Y 2 receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H 2O 2 induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y 2 cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y 2 receptor.

Bisphosphonates activate nucleotide receptors signaling and induce the expression of Hsp90 in osteoblast-like cell lines

Bisphosphonates are the most important drugs used in the treatment of osteoporosis as they inhibit osteoclast resorption and stimulate proliferation of osteoblasts. However, the molecular mechanisms responsible for these effects are still poorly elucidated. It is known that nucleotide receptors-mediated signaling plays a central role in modulating osteoblasts growth in response to mechanical stress. By using osteoblast-like cell lines (i.e., HOBIT, MG-63, ROS P2Y), which express P2Y receptors, we found that the treatment with risedronate promotes non-lytic ATP release leading to activation of ERKs through the involvement of P2Y receptors triggering. A major role in this signal transduction pathway seems to be the involvement of P2Y 1 and P2Y 2 receptors, since the stimulatory effect of risedronate on ERKs is not appreciable in ROS 17/2.8 cells, which do not express these two receptors. Differential proteomics analysis identified Hsp90 upregulation as a result of risedronate effect on HOBIT and MG-63 cells. The stimulatory effect is dependent on ERKs activation involving nucleotide receptors triggering and leads to increased proliferation of osteoblast-like cells. In fact, functional inactivation of Hsp90 by the specific inhibitor 17-AAG prevents the bisphosphonate-induced mitogenic effects in osteoblasts. These findings show that bisphosphonates, by inducing ATP release, may also act through nucleotide receptors signaling leading to ERKs activation and may exert their mitogenic role on osteoblasts through the involvement of Hsp90.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: Evidence for an autoregulatory loop

The Early Growth Response protein (Egr-1) is a C2H2–zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation–reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H2O2 stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H2O2 stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Osteoblasts induce prostate cancer proliferation and PSA expression through interleukin-6-mediated activation of the androgen receptor

Prostate cancer (CaP) metastases selectively develop in bone as opposed to other sites through unknown mechanisms. Interleukin-6 (IL-6) is considered to contribute to CaP progression and is produced at high levels in osteoblasts. We hypothesized that osteoblast-derived IL-6 in the bone microenvironment contributes to the fertile soil for CaP growth. Accordingly, human CaP cells, LNCaP, C4-2B and VCaP, were treated with conditioned medium (CM) collected from human osteoblast-like HOBIT cells grown in androgen-depleted medium. We found that CM induced proliferation, prostate-specific antigen (PSA) protein and mRNA expression in a dose-dependent manner in these cell lines as determined by ELISA and real-time PCR, respectively. CM also activated the PSA promoter in these cells. Both HOBIT and primary osteoblast (POB) cells produced high levels of IL-6 measured by bioassay. LNCaP, C4-2B and VCaP cells expressed IL-6, but at much lower levels then the HOBIT and POB and they also expressed the IL-6 receptor mRNA, indicating they can respond to IL-6. Anti-IL-6 antibody added to HOBIT or POB CM dose-dependently inhibited the CM-induced cell proliferation and PSA expression in these CaP cell lines. HOBIT CM induced nuclear translocation of the AR and this was inhibited by anti-IL-6 antibody. Additionally, the antiandrogen bicalutamide inhibited HOBIT CM-induced cell proliferation. These results demonstrate that osteoblasts promote CaP growth through IL-6-mediated activation of the AR. Furthermore, these data underscore the importance of cross-talk between tumor and the bone microenvironment in the development of CaP bone metastases.

Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line.

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y(1) and P2Y(2) G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C(2)H(2)-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca(2+)-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.

Extracellular NAD +: a novel autocrine/paracrine signal in osteoblast physiology

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD + is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD + with oscillatory increases of the cytosolic Ca 2+ concentration. The initial Ca 2+ response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD + in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD + as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.

Mechanically Induced ATP Release from Human Osteoblastic Cells

Extracellular ATP is a widespread autocrine/paracrine signal since many animal cells release ATP in the extracellular medium; often this release is mechanosensitive, but the molecular mechanism is still unclear. The involvement of vesicular release, conductive channels, or ABC transporters has been suggested in different cell types. We investigated the mechanism of ATP release in human HOBIT osteoblastic cells, in which mechanical stimulation induced intercellular calcium waves sustained by both cell-to-cell coupling through gap junctions and ATP release. In this study we employed a luciferin-luciferase bioluminescence assay to measure the amount of ATP released under different stimulatory conditions. Given the role of connexin hemichannels in favoring passive NAD+ transport [Bruzzone, S., et al. (2001) FASEB J. 15, 10–12], the involvement of connexin hemichannels as putative ATP transporters was initially investigated. In HOBIT cells overexpressing connexin43 the amount of nucleotide released under basal and stimulated conditions was similar to non-transfected cells, ruling out a major involvement of connexin hemichannels in ATP transport. In nontransfected HOBIT cells mechanical stimulations induced by medium displacement and hypotonic stress consistently enhanced ATP efflux. Cytochalsin D treatment did not alter basal and stimulated ATP release, while elevated cAMP levels consistently reduced efflux in both cases. ATP released by hypotonic stress and medium displacement evoked intracellular Ca2+ transients in fura2-loaded HOBIT cells, indicating that different mechanical stimuli activate physiological cell responses.

Ethanol activates NFκB DNA binding and p56 lck protein tyrosine kinase in human osteoblast-like cells

Alcoholics frequently suffer from moderate to severe bone loss that results in bone fractures. Both decreased bone production and increased bone resorption have been postulated to contribute to ethanol (ETOH)-mediated bone loss. Bone resorption is induced by several proinflammatory cytokines such as interleukin-1 and -6. The expression of these cytokines is induced by the transcription factor NFκB, which, in turn, is activated by several kinases. It follows that protein kinase and NFκB activation may contribute to ETOH-induced bone loss. Accordingly, we sought to determine if ETOH activates protein tyrosine kinases (PTK) and NFκB DNA binding in a human osteoblast-like cell line (HOBIT). Ethanol at 50 and 100 mmol/L (reflective of blood ethanol levels reached in chronic alcoholics) for 24 h did not alter HOBIT cell viability. In contrast, 200 mmol/L ethanol decreased cell viability by 40%. Treatment of HOBIT cells with 100 mmol/L ETOH induced nuclear NFκB:DNA complex formation and NFκB activity. Incubation of HOBIT cells with ETOH at 50 and 100 mmol/L for 30 min induced a 2.5- and 4.2-fold increase in PTK activity, respectively. Preincubation of HOBIT cells with damnacanthal (DAM), which inhibits p56 lck , blocked ETOH-mediated PTK activity; whereas, preincubation with herbimycin A, which inhibits pp60 src , did not. DAM inhibited both ethanol-induced NFκB activation in HOBIT cells and interleukin-6 expression in primary human osteoblasts. Finally, preincubation with the protein kinase C inhibitor, bisindolylmaleimide I HCl (BIS), diminished ETOH-mediated PTK activity; whereas, preincubation with the protein kinase A inhibitor, H89, did not. These data demonstrate that ETOH induces NFκB nuclear translocation through p56 lck in HOBIT cells. BIS′ inhibition of PTK activation suggests that ETOH activates PTK through a protein kinase C-dependent pathway. These data suggest that ETOH may contribute to bone loss through activation of signal transduction that results in production of an osteoclastogenic cytokine (i.e., interleukin-6) in osteoblasts.

Ethanol and Acetaldehyde Inhibit the Formation of Early Osteoblast Progenitors in Murine and Human Bone Marrow Cultures

Alcohol abuse is commonly associated with reduced bone mass and osteoporosis as a consequence of both systemic and direct cellular effects. To clarify some of the pathways by which alcohol exerts its actions directly on bone cells, we investigated the formation of early osteoblast progenitors (colony-forming units for fibroblasts; CFU-F) in long-term murine and human bone marrow cultures exposed to ethanol and to its main metabolite, acetaldehyde. In murine bone marrow cultures, obtained from Swiss female mice, ethanol inhibited CFU-F formation (maximal reduction +/- SEM: 50 +/- 2%; p < 0.01) at concentrations ranging from 0.04% to 0.6% that are similar to those reached in vivo in alcoholics. Acetaldehyde strongly reduced CFU-F formation at concentrations of 0.004% and 0.02%, and completely abolished it at the dose of 0.06%. Similarly, ethanol (at concentrations > or =0.02%) and acetaldehyde (from 0.004% to 0.06%) significantly decreased the number of CFU-F in human bone marrow cultures; the mean reduction observed with ethanol was 63 +/- 12% (p < 0.05), whereas acetaldehyde completely prevented CFU-F formation at the concentration of 0.06%. These in vitro observations were confirmed by the in vivo findings that the CFU-F formation in bone marrow cultures from nine young, chronic, noncirrhotic alcoholics was significantly reduced (70 +/- 15%), compared with seven age-matched normal subjects (p < 0.01). In addition, acetaldehyde inhibited cell proliferation in human osteoblastic cells (MG-63 and HOBIT cell lines), whereas ethanol reduced proliferation only in MG-63 cells. Our results indicate that ethanol and acetaldehyde may directly inhibit the osteoblastogenic potential of the bone marrow, and this effect may contribute to the decreased bone formation observed in alcoholics.

Normal human osteoblast-like cells consistently express genes for insulin-like growth factors I and II but transformed human osteoblast cell lines do not.

Insulin-like growth factors I (IGF-I) and II (IGF-II) are anabolic for osteoblastic cells. Although expression of IGF-I and IGF-II mRNA has been demonstrated in rodent osteoblastic cells, little is known about IGF gene expression in human osteoblastic cell models. In this study we characterized IGF-I and -II mRNA expression in (1) normal human osteoblast-like (hOB) cells, (2) a simian virus 40 immortalized hOB (HOBIT) cell line, and (3) human osteosarcoma cell lines SaOS-2, TE-85, MG-63, and U-2. Since cross-hybridization of IGF cDNA probes with ribosomal RNA obscures detection of some of the multiple IGF transcripts in human cells, we replaced Northern analysis with the more specific ribonuclease protection assay (RPA). We also used the reverse transcriptase-polymerase chain reaction (RT-PCR) to assess whether mRNAs were present at trace levels. IGF-I mRNA expression was consistently observed in normal hOB cells only and by both RT-PCR and RPA. Among IGF-I transcript variants, Ea IGF-I mRNA was more abundant than the Eb mRNA in normal hOB cells. Trace levels of IGF-I mRNA were variably detected in SaOS-2 and U-2 osteosarcoma cells when RT-PCR was performed, but we found no IGF-I mRNA in HOBIT, TE-85, or MG-63 cells. IGF-II mRNA was expressed in normal hOB, HOBIT, TE-85, and U-2 cells as assessed by either method.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in insulin-like growth factor (IGF)-dependent IGF-binding protein-4 proteolysis in transformed osteoblastic cells.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is secreted by a variety of osteoblastic cells and appears to be an integral component of bone cell physiology. We have previously reported that normal human osteoblast-like (hOB) cells secrete IGFBP-4 as well as a novel IGFBP-4 protease, which requires IGF for functional activity. In this study we assessed the IGFBP-4/IGFBP-4 protease system in transformed osteoblastic cells by Western ligand blotting and cell-free IGFBP-4 protease assays. Simian virus-40-immortalized hOB cells (HOBIT), human osteosarcoma cells (TE-85), and rat osteosarcoma cells (UMR 106-01, ROS 17/2.8) secrete IGFBP-4. In contrast to the rapid and dramatic proteolysis in hOB medium, medium conditioned by these cells had no apparent IGFBP-4 protease activity when assayed with exogenous IGF-II in culture or under cell-free conditions. Assayed in the presence of exogenous protease. HOBIT cells, but not the osteosarcoma cell lines, appeared to produce a cycloheximide-sensitive inhibitor of the IGFBP-4 proteolytic reaction. Transient cell transformation induced by incubating human osteoblasts transfected with a temperature-sensitive mutant of simian virus-40 T-antigen at the permissive temperature or by treating hOB cells with phorbol ester tumor promoters also resulted in inhibition of IGF-dependent IGFBP-4 proteolysis. Inhibition was observed if phorbol ester was added to the cultures at the time of medium change or after the protease had been expressed and secreted. Differences in IGFBP-4 proteolysis could not be accounted for by changes in IGFBP-4 messenger RNA expression or substrate levels. These data suggest that transformation is associated with alterations in the IGFBP-4/IGFBP-4 protease system in osteoblastic cells. Normal human osteoblasts secrete an IGF-dependent IGFBP-4 protease. The induction of an inhibitor of the IGF-dependent IGFBP-4 proteolytic reaction may be associated with early transformation processes. Fully tumorigenic bone cells expressed neither IGFBP-4 protease nor protease inhibitor activity.

Sex hormones mediate interleukin-1β production by human osteoblastic HOBIT cells

The mechanisms by which the sex hormones achieve their bone-sparing effects remains unresolved. Interleukin-1β (IL-1β) is an autocrine/paracrine regulator of bone that may be produced in an estrogen-sensitive manner. The regulation of IL-1β production by the gonadal steroids was tested in the human osteoblastic HOBIT cell model. Dose-dependent 4–8-fold increases ( P < 0.05) in IL-1β mRNA levels followed a 6–48 h treatment with 17β-estradiol or testosterone. Receptor mediation of these responses was indicated by experiments using 17α-estradiol or flutamide. Tumor necrosis factor-α (TNF) dependent increases in IL-1β mRNA levels were additive to the effects of the steroids. Testosterone and TNF increased IL-1β protein release ( P < 0.05) while 17β-estradiol had little effect on release. The bone-sparing effects of the gonadal steroids may be accomplished, in part, through their mediation of local IL-1β production.

Recombinant human bone morphogenetic protein‐2 enhances expression of interleukin‐6 and transforming growth factor‐β1 genes in normal human osteoblast‐like cells

The process of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced endochondral ossification involves 1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; 2) the production and maturation of cartilage and bone matrix; and 3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP-2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast-like initial transfectant cell line (HOBIT) to study the effect of rhBMP-2 on gene expression of interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-beta 1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP-2 acts on HOBIT cells to stimulate expression of IL-6 and TGF-beta 1 genes and the production of IL-6. Enhancement of gene expression of IL-6 and TGF-beta 1 by rhBMP-2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF-beta 1 and IL-6 mRNA occurs within short periods--4 and 8 hours after exposure to rhBMP-2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP-2. It suggests that rhBMP-2 enhances IL-6 and TGF-beta 1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP-2-induced endochondral ossification.