Osteoactivin Expression Research Articles

Cycloastragenol prevents age-related bone loss: Evidence in d-galactose-treated and aged rats

Background and aimsAging-induced bone loss is a multifactorial, age-related, and progressive phenomenon among the general population and may further progress to osteoporosis and increase the risk of fractures. Cycloastragenol (CAG), currently the only compound reported that activates human telomerase, is thought to be able to alleviate or delay the symptoms of aging and chronic diseases. Previous research has suggested that CAG may have the potential to alleviate age-related bone loss. However, to date, no research has specifically focused on this aspect. In this study, we aimed to investigate whether CAG could prevent senile osteoporosis, and further reveal its underlying mechanism. MethodsCAG treatment was administrated into two bone loss rat models (D-galactose administration and aging) for 20 weeks and 33 weeks, respectively. Serum biomarkers analyses, bone biomechanical tests, micro-computed tomography assessment, and bone histomorphometry analyses were performed on the bone samples collected at the endpoint, to determine whether CAG could prevent or alleviate age-related bone loss. Proteomic analysis was performed to reveal the changes in protein profiles of the bones, and western blot was used to further verify the identity of the key proteins. The viability, osteoblastic differentiation, and mineralization of MC3T3-E1 cells were also evaluated after CAG treatment in vitro. ResultsThe results suggest that CAG treatment improves bone formation, reduces osteoclast number, alleviates the degradation of bone microstructure, and enhances bone biomechanical properties in both d-galactose- and aging-induced bone loss models. CAG treatment promotes viability, osteoblastic differentiation, and mineralization in MC3T3-E1 cells. Proteomic and western blot analyses revealed that CAG treatment increases osteoactivin (OA) expression to alleviate bone loss. ConclusionThe results revealed that CAG alleviates age-related bone loss and improves bone microstructure and biomechanical properties. This may due to CAG-induced increase in OA expression. In addition, the results support preclinical investigations of CAG as a potential therapeutic medicine for the treatment of senile osteoporosis.

Growth and repair factors, osteoactivin, matrix metalloproteinase and heat shock protein 72, increase with resolution of inflammation in musculotendinous tissues in a rat model of repetitive grasping.

BackgroundExpression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats.MethodsFlexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines.ResultsFlexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6.ConclusionThe simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.

Inhibition of PI3K/Akt and Erk Signaling Pathways by BCR-ABL Tyrosine Kinase Inhibitors up-Regulates the Immunosuppressive Receptor Osteoactivin in Monocyte-Derived Dendritic Cells

AbstractAbstract 1050Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is expressed in dendritic cells (DC). Osteoactivin/syndecan-4 (SD-4) interaction was previously shown to inhibit T cell activation by antigen-presenting cells. We recently demonstrated that exposure of human peripheral blood monocytes to IL-10 or pharmacological levels of the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib or nilotinib during differentiation into monocyte-derived DC (moDC) causes up-regulation of osteoactivin at the transcript and protein level in vitro (Schwarzbich et al., 2012). Here we aimed to elucidate the molecular mechanisms responsible for osteoactivin up-regulation in moDC upon exposure to IL-10 or TKI (imatinib, nilotinib). moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Every second day, starting from the first day of culture, IL-10 or TKI were added to the culture medium. Alternatively, specific inhibitors of PI3K, Akt, Erk and/or C-Raf signal transduction pathways were added to the cell cultures. Cells were harvested on day 7 of culture for immunophenotyping and osteoactivin expression analysis using FACS, western-blotting and real-time qRT-PCR. The expression and activity of signal transduction molecules was monitored by western-blotting and pathway protein arrays. Analysis of moDC function was performed using mixed lymphocyte reactions (MLR). Our in vitro analysis revealed that IL-10 and BCR-ABL TKI inhibit the PI3K/Akt and, to a lesser extent, the Erk pathway in moDC. Specific inhibition of these signal transduction cascades resulted in profound up-regulation of osteoactivin expression in moDC confirming the involvement of PI3K/Akt and Erk in the regulation of osteoactivin expression. In line, TKI treatment and inhibition of PI3K/Akt and Erk comparably affected the phenotype of moDC. Moreover, the inhibition of these signalling cascades resulted in reduced stimulatory capacity of moDC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. Our data reveal that TKI exert immunosuppressive effects in moDC by interfering with pathways involved in IL-10 receptor signaling. Inhibition of osteoactivin expression or function may thus constitute a promising strategy in combinatory approaches using TKI and DC-based immunotherapy and may enhance the efficacy of immunotherapeutic interventions in cancer patients. Disclosures:No relevant conflicts of interest to declare.

Upregulation of Osteoactivin Upon Exposure to Tyrosine Kinase Inhibitors Impairs the T Cell Stimulatory Capacity of Monocyte-Derived Dendritic Cells

Abstract 1112▪▪This icon denotes a clinically relevant abstractTargeted therapies using tyrosine kinase (TK) inhibitors have significantly improved the treatment of cancer patients. Imatinib (Glivec, Gleevec, STI 571) was the first TK inhibitor (TKI) established for the treatment of cancer and efficiently blocks the activity of c-ABL, a non-receptor TK which is pathologically activated in philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML). Nilotinib (Tasigna) and dasatinib (Sprycel) are second-generation TKI that have shown efficacy in the treatment of Ph+ CML resistant or intolerant to imatinib. However, molecularly detectable disease persists in the majority of patients treated with TKI, causing relapse after discontinuation of TKI treatment in many cases. Thus, multiple approaches presently aim to combine TKI treatment with immunotherapy. As TKI, besides targeting their eponymous enzyme, influence multiple other signaling pathways involved in cellular functions, analysis of potential effects of TKI on immune effector cells may be key to develop successful combinatorial strategies. Due to their unique ability to initiate powerful anti-tumor T cell responses, dendritic cells (DC) are employed in many immunotherapeutic strategies aiming to eradicate the malignant cell population. Upon activation they change their expression pattern of cell surface molecules and secreted cytokines/chemokines, a process called DC maturation. Osteoactivin, also known as transmembrane glycoprotein NMB (GPNMB) and dendritic cell-associated transmembrane protein (DC-HIL), is a type I transmembrane glycoprotein that is detected abundantly in DC but not or substantially less in monocytes. Its expression can inhibit T cell activation by binding the type 1 transmembrane proteoglycan syndecan-4 (SD-4) on T cells. Here we extend our findings that the exposure of human peripheral blood monocytes to the immunosuppressive and anti-inflammatory cytokine IL-10 or to therapeutic concentrations of TKI during differentiation into monocyte-derived DC (moDC) leads to significant upregulation of osteoactivin at the transcript and protein level in vitro (Blood 2010 116: abstract 1733). We analyzed the expression of other inhibitory receptors, such as PD-L1, PD-L2, CD80, or CD86 and observed no significant differences of the expression under TKI treatment. Furthermore, we thoroughly examined the expression of osteoactivin in the presence of relevant maturation signals such as TLR ligands, IFN-γ or TNF. LPS, Poly I:C, Pam3Cys or R848 nearly abolished osteoactivin expression compared to untreated control cells. In contrast, IFN-γ or TNF did not significantly reduce osteoactivin expression below the basal level. To evaluate the involvement of osteoactivin in TKI-triggered effects on moDC function we performed mixed lymphocyte reactions with allogenic T cells. Osteoactivin upregulation upon exposure to imatinib, dasatinib and nilotinib resulted in significantly reduced T cell stimulatory capacity of moDC. This was not due to IL-10 upregulation but rather due to direct inhibitory effects of osteoactivin on T cell proliferation which could be overcome by addition of blocking anti-osteoactivin antibody. Our data demonstrate that upregulation of osteoactivin upon exposure of immature moDC to TKI is critically involved in the inhibition of DC function. These findings indicate that inhibition of osteoactivin expression or function may serve as a novel strategy in combinatory approaches using TKI and DC-based immunotherapy and may enhance the efficacy of immunotherapeutic interventions in cancer patients. Disclosures:No relevant conflicts of interest to declare.

Immunosuppressive Drugs Upregulate the Immunoinhibitory Receptor Osteoactivin in Monocyte-Derived Dendritic Cells,

Abstract 3232Osteoactivin, also known as transmembrane glycoprotein NMB (GPNMB) and dendritic cell-associated transmembrane protein (DC-HIL), is a type I transmembrane glycoprotein that is detected abundantly in dendritic cells (DC), but not or only at low levels in monocytes. Its expression on antigen-presenting cells (APC) can inhibit T cell activation by binding the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells. We here studied the influence of different therapeutically used immunosuppressive agents such as the corticosteroid prednisolone and the calcineurin inhibitors tacrolimus or cyclosporine A on the expression of osteoactivin in human monocyte-derived DC. Therefore, DC were generated from blood monocytes isolated by plastic adherence and exposed to GM-CSF and IL-4. Prednisolone, tacrolimus or cyclosporine A were added to the culture medium at concentrations obtained upon clinical application in patients (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml) every second day starting from the first day of culture. Cells were harvested for immunophenotyping and osteoactivin expression analysis by immunostaining, western-blotting and real-time RT-PCR or for functional analyses in mixed lymphocyte reactions (MLR) on day 7 of culture. DC generated in the presence of therapeutic concentrations of the immunosuppressant drugs showed a reduced expression of CD1a. Furthermore, we observed a clear upregulation of osteoactivin on DC that had been treated with the immunosuppressive agents. In line with osteoactivin up-regulation, exposure to the immunosuppressive drugs resulted in reduced stimulatory capacity of the DC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. In summary, our results demonstrate that upregulation of osteoactivin is critically involved in the inhibition of DC function upon exposure to different classes of immunosuppressive agents. These findings suggest that upregulation of osteoactivin in DC constitutes a ubiquitous mechanism that may underlie inhibition of T cell activation by these drugs. Disclosures:No relevant conflicts of interest to declare.

The Immune-Inhibitory Receptor Osteoactivin Is up-Regulated In Monocyte-Derived Dendritic Cells Upon Exposure to Tyrosine Kinase Inhibitors

Abstract Abstract 1733 Targeted therapies with tyrosine kinase inhibitors (TKI) have significantly improved the treatment of cancer patients. Ex vivo generated dendritic cells (DC) are commonly used in immunotherapeutic strategies due to their unique ability to initiate adaptive immune responses, and multiple approaches presently aim to combine targeted therapies with immunotherapy. However, as many kinases targeted by TKI are, besides governing tumor cell growth, also involved in the activation of DC, TKI therapy may cause immunoinhibitory side effects. Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is detected abundantly in DC but not in monocytes. Its expression on antigen-presenting cells can inhibit T cell activation by binding syndecan-4 (SD-4) on T cells. Here we investigated the effect of the BCR/ABL TKI imatinib, dasatinib and nilotinib, which are approved for the treatment of CML, on the expression of osteoactivin and DC functions. DC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Imatinib, nilotinib or dasatinib were added to the culture medium every second day starting from the first day of culture. In some experiments, toll-like receptor (TLR) ligands (L) (LPS (TLR4L), pam3Cys (TLR2L), poly I:C (TLR3L) or R848 (TLR7/8L) were added on day 6 of culture for maturation of DC. We found that DC generated in the presence of therapeutic concentrations of all three TKI displayed an altered phenotype. Imatinib caused significantly reduced expression of the typical DC markers CD1a, CD83 and the co-stimulatory molecule CD86. Nilotinib reduced the expression of CD1a, CD83, CD86 and the DC-specific C-type lectin receptor DC-SIGN (CD209). Dasatinib impaired expression of CD1a, CD83, CD86, CD80 and DC-SIGN. Most notably, we observed excessive up-regulation of osteoactivin on DC upon treatment with all three TKI. Interestingly, incubation with the immunosuppressive and anti-inflammatory cytokine IL-10 also resulted in osteoactivin over-expression. In line with osteoactivin up-regulation, exposure to TKI resulted in reduced stimulatory capacity of DC in MLR with allogenic T cells that could be restored by addition of blocking anti-osteoactivin antibody. In summary, our data demonstrate that up-regulation of osteoactivin is critically involved in the inhibition of DC function upon TKI exposure. These findings are of great importance for future combinatory approaches using TKI and DC-based immunotherapy and indicate that inhibition of osteoactivin expression or function may serve as a novel strategy to enhance the efficacy of immunotherapeutic interventions in cancer patients. Disclosures: No relevant conflicts of interest to declare.

Molecular Regulation of Osteoactivin Expression in Osteoblasts

Osteoblast (OB) commitment, proliferation and differentiation from mesenchymal stem cells are regulated by Bone Morphogenetic Protein2 (BMP2). BMP2 mediates these osteoinductive effects by recruiting Transcription Factors (TFs), namely Runx2, Homeodomain (HD), Smad1 and Smad4 proteins to the promoter of OB specific genes and induces their transcription. Our laboratory has previously shown that Osteoactivin (OA), OB‐related glycoprotein acts as a downstream mediator of BMP2 effects on OB differentiation and function. Furthermore, we showed that Smad1 signaling regulates BMP2 induced OA expression. The goal of our study is to characterize OB specific TFs that regulate BMP2 induced OA promoter activity. In order to study the effects of BMP2 on OA promoter activity, we cloned 1kb upstream of rat OA gene into a luciferase expression vector and have shown that BMP2 stimulates OA promoter activity in a time and dose‐dependent manner. Secondly, knockdown of Runx2, HD, Smad1 and Smad4 using specific siRNAs decreased BMP2 induced OA promoter activity. Similarly, loss of consensus binding sites of these TFs from OA promoter decreased OA promoter activity. Furthermore, ChiP assays at various stages of OB differentiation indicated that Runx2, Smad1, Smad4 and HD proteins associate differentially with OA promoter. Collectively, our results suggest that Runx2, Smad1, Smad4 and HD TFs differentially regulate BMP2 induced OA promoter activity during OB differentiation.Grant Funding Source: NIAMS

Osteoactivin/HGFIN: is it a tumor suppressor or mediator of metastasis in breast cancer?

In the previous issue of Breast Cancer Research, Metz and colleagues characterize hematopoietic growth factor inducible neurokinin-1 type (HGFIN; osteoactivin, Gpnmb, Dc-HIL) as a gene expressed in normal mammary epithelial cells that encodes a protein with tumor suppressor functions in breast cancer [1]. In contrast, we have recently identified osteo-activin as a novel mediator promoting breast cancer metastasis [2] and thus feel it is premature to classify osteoactivin as a tumor suppressor in breast cancer. In their article, Metz et al. claim that osteoactivin expression is detectable in non-tumorigenic cells, but low to undetectable in malignant This conclusion was based on RNA expression in three breast cell lines and three primary breast tissues. In our study, we analyzed microarray data from a recently published data set [3] that examined gene expression profiles of three non-malignant and 51 malignant human breast cell lines. We confirmed these microarray data by performing quantitative real-time polymerase chain reaction (PCR) of osteoactivin expression in nine of these cell lines [2]. Our analysis revealed that 48 of the 51 breast cancer cell lines examined possessed higher osteoactivin expression than normal breast MCF-12A cells. In contrast to the data from Metz et al., the T47D and HCC70 breast cancer cell lines possessed 2.4 and 48.5 fold higher osteoactivin expression, respectively, relative to MCF-12A cells. The authors also performed in situ hybridization for osteo-activin mRNA in paraffin embedded breast biopsies. They conclude that osteoactivin is found at higher levels in benign tissues than in malignant tissues, and that no correlation exists between osteoactivin expression and estrogen receptor (ER) status. It must be noted that their conclusions are in contrast to several published large scale analyses of primary human breast tumors that are available through the online cancer microarray database, Oncomine [4]. These studies indicate that osteoactivin expression is significantly higher in both breast cancer versus normal breast specimens and in ER negative versus ER positive breast tumors [2]. Metz et al. state: Osteoactivin is expressed in breast cancer cells and has been associated with bone invasion, and aggressive form of the tumor. They repeatedly refer to Rich et al. to corroborate this remark, however, this particular reference does not provide any evidence to support this statement [5]. Rich et al. were, in fact, investigating a role for osteoactivin in promoting the invasion of a defined glioma model. This study did not employ breast cancer cells in any experiments, nor was bone metastasis investigated. In our study, we report, for the first time, that ectopic osteoactivin expression in breast cancer cells is sufficient to induce a significant increase in the incidence of osteolytic bone metastases in vivo. Finally, Metz et al. refer to a study that links osteoactivin expression to the low metastatic potential of melanoma cell lines to support the notion that osteoactivin functions as a tumor suppressor [6]. However, the authors have overlooked more recent studies that identify osteoactivin as being highly expressed in metastatic melanoma-derived cell lines and clinical specimens. These studies report the development of a cytotoxin-conjugated, anti-osteoactivin antibody (CR011-vcMMAE) that is currently in Phase I/II clinical trials as a treatment for patients with Stage III and Stage IV melanoma [7,8]. In summary, osteoactivin is emerging as an important, yet controversial effector of tumor progression in several cancer types, including breast. As such, the pursuit of rigorous, functional studies – aimed at addressing the mechanisms of osteoactivin-mediated effects on tumor progression and metastasis in vivo – will be critical to assess its feasibility as a target for therapeutic intervention in breast cancer.

Osteoactivin Promotes Breast Cancer Metastasis to Bone

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor-negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone.

Early induction of osteoactivin expression in rat renal tubular epithelial cells after unilateral ureteral obstruction

In this study, we examined the expression of osteoactivin in the rat kidney after unilateral ureteral obstruction. Male Wistar rats were sacrificed at 6 h, and on days 1, 2, 3 and 7 after the obstruction. The renal tubular lumens gradually dilated, and marked interstitial fibrosis was confirmed histologically on day 3 after the obstruction. The expressions of osteoactivin and collagen type III were examined by quantitative real-time RT-PCR. An 8-fold increase in osteoactivin mRNA expression as compared with that in the sham-operated group was observed at 6 h after the obstruction, whereas no elevation of collagen type III mRNA expression was observed at this early stage. Furthermore, semi-quantitative RT-PCR was performed to identify upregulation of expression of matrix metalloproteinase-13 mRNA relative to that in the sham-operated control, and normalized to the expression level of β-actin. Intense osteoactivin expression localized in the dilated tubular epithelium and interstitial fibroblasts in the obstructive kidney was detected by immunohistochemistry and by in situ hybridization. These results suggested that the early-phase upregulation of osteoactivin expression in the tubular epithelium in response to renal injury might play a role in triggering renal interstitial fibrosis via activation of matrix metalloproteinase expression and collagen remodeling in rats.

Expression of osteoactivin in rat and human liver and isolated rat liver cells

The transmembrane glycoprotein osteoactivin is expressed in osteoblasts, dendritic cells and tumor cells. It is suggested to influence osteoblast maturation, cell adhesion and migration. We studied osteoactivin expression within the liver. Expression of osteoactivin was analysed by RT-PCR, Northern blotting, in situ hybridisation (ISH) and immunohistochemistry (IHC) comparing normal and acutely injured rat liver [induced by carbon tetrachloride (CCl(4)) administration], liver cell populations, and normal or diseased human liver. By ISH osteoactivin expression was detected in sinusoid-lining cells found by IHC to be positive for the common mononuclear phagocyte marker antibody anti-CD68. While total liver contained only traces, isolated Kupffer cells expressed abundant amounts of osteoactivin mRNA further increasing during culture. In acutely injured rat liver osteoactivin expression was strongly increased reaching maximum of expression 48h after CCl(4). By ISH osteoactivin expression was localised in pericentral inflammatory cells and sinusoid-lining cells again anti-CD68 positive. Dexamethasone and lipopolysaccharide decreased osteoactivin expression in cultured mononuclear phagocytes of normal as well as of acutely injured liver. In human liver osteoactivin was increased in fulminant hepatitis and paracetamol intoxication. Osteoactivin is expressed at high levels in normal and inflammatory liver macrophages suggesting a significant role in acute liver injury.

Expression of the novel gene osteoactivin in rat and human liver tissue and isolated liver mononuclear phagocytes

The transmembrane glycoprotein osteoactivin is found in osteoblasts, dendritic cells and tumor cells and is supposed to plays a role in osteoblast maturation and cell adhesion. Methods: Total RNA was extracted from rat organs, and from cultured liver cell populations (hepatocytes, Kupffer cells, sinusoidal endothelial cells, hepatic stellate cells, myofibroblasts), and analyzed by Northern blot hybridization. Moreover, inflammatory mononuclear phagocytes (MNP) were isolated from carbon tetrachloride (CCl4)-treated rats. Cultured Kupffer cells and MNP were treated with lipopolysaccharide (LPS) or dexamethasone (DEX). Total RNA was extracted from human liver (acute liver failure, liver cirrhosis from different origin). Results: Organ expression of osteoactivin showed high levels in lungs and spleen, less in kidney and colon. Trace osteoactivin expression was found in normal rat liver. High levels of osteoactivin mRNA were detected in normal Kupffer cells. LPS and DEX treatment of Kupffer cells led to a decrease of osteoactivin mRNA. In CCl4-treated animals osteoactivin mRNA was abundantly detected in total liver RNA and in isolated inflammatory MNP of the liver. DEX treatment of animals and of cultured inflammatory MNP decreased osteoactivin mRNA expression. Human liver total RNA extracts contained elevated osteoactivin mRNA levels in fulminant hepatitis. Conclusions: This is the first description of abundant osteoactivin expression in Kupffer cells as well as in liver inflammatory MNP. The inhibitory effect of glucocorticoids suggests a role of osteoactivin in inflammatory processes.

Bone-related Genes Expressed in Advanced Malignancies Induce Invasion and Metastasis in a Genetically Defined Human Cancer Model

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitro phenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis.

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.