Osteopontin Stimulation Research Articles

OP24 SPP1 in colitis-associated colon cancer

Abstract Background Patients with ulcerative colitis (UC) and Crohn’s disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analyses. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis, utilizing human samples from patients with CD, CD-associated CAC (CD-CAC), UC, and UC-associated CAC (UC-CAC). Findings were further validated in vitro using human colon organoids. Methods A Nanostring Immunology v2 panel was employed on RNA (archival FFPE sections) from 40 patients suffering from either IBD or CAC, alongside inflammation-free healthy controls (n=10 per group). Our data revealed a robust upregulation of the SPP1 (Secreted Phosphoprotein 1) gene encoding osteopontin (OPN) in CAC patients. Prospectively, we extended our methodology to an unbiased imaging-based spatial RNA in situ sequencing (ISS) approach (477 genes, Xenium, 10X Genomics) using FFPE sections from CAC and UC patients (n=3 per group). This provided insights into the spatial expression of SPP1 and its target receptors at a subcellular resolution. Findings were validated using whole FFPE section confocal microscopy. In addition, in vitro models, including human UC organoids, were analyzed by bulk RNA-Seq, RT-qPCR, and downstream functional assays. Results Nanostring Immunology v2 gene expression data revealed upregulation of the SPP1 gene encoding OPN (Fig 1A) and EMT transcription factors FN1 and ZEB1 in CAC patients. Xenium RNA in situ sequencing (ISS) unveiled the spatial heterogeneity of SPP1 expression in CAC (Fig 1B). Unsupervised graph-based clustering identified an SPP1+CD68+ macrophage cluster exclusively in CAC (Fig 1C), validated through OPN/CD68 immunostainings (Fig 1D). In vitro OPN stimulation across multiple human and mice colon organoid models excluded OPN as the trigger of EMT. Interestingly, Xenium ISS and immunostainings revealed a mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells (Fig 1E, F and G). Conclusion SPP1 is significantly upregulated in CAC (RNA and protein), expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. The mutually exclusive spatial locations of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial role of SPP1/OPN in modulating the immunosurveillance of CAC cells by CD8+ tumour lymphocytes.

Osteopontin in the Pathogenesis of Aortic Dissection by the Enhancement of MMP Expressions.

The pathogenesis of aortic dissection (AD) is unclear. The aim of this study was to explore the relationship between osteopontin (OPN) and AD. Fifty AD patients were enrolled; 29 had hypertension with AD (H-AD) and 21 no hypertension with HD (NH-AD). Twenty-five healthy controls (NH-C) and 14 patients with hypertension (H-C) were also enrolled. Serum and aortic wall OPN levels were determined. Human vascular muscle cells (HVSMC) were stimulated by both low (1 μg/mL) and high (5 μg/mL) concentrations of OPN and cell proliferation as well as apoptosis was measured. Transforming growth factor-β (TGF-β), matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-9, TIMP-1, and TIMP-2 gene expressions by HVSMC were measured and Akt, IκB, Smad1/5/8 and Erk1/2 signaling pathways were detected. Our results showed that AD patients demonstrated significantly higher levels of serum and local OPN expressions compared to healthy controls. In those with hypertension, the serum concentrations of OPN were increased compared to those without hypertension. In in vitro culture, a high dose of OPN stimulation promoted the proliferation of HVSMC but did not affect cell apoptosis. Both concentrations of OPN enhanced MMP-2 gene expression and its activity in HVSMC. Moreover, Akt and IκB signaling pathways were significantly activated after OPN stimulation while the Smad1/5/8 and Erk1/2 signaling pathways were not changed. The addition of an IκB inhibitor significantly abrogated MMP-2 gene expression. Our data show that OPN may participate in the pathogenesis of AD by the enhancement of MMP-2 expression.

Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration

Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses.

Osteopontin induces vascular endothelial growth factor expression in articular cartilage through PI3K/AKT and ERK1/2 signaling.

The expression of osteopontin (OPN) and vascular endothelial growth factor (VEGF) are associated with the severity of cartilage destruction in osteoarthritis. However, the biological connection between OPN and VEGF in osteoarthritis remains to be elucidated. The present study was performed to investigate the effect of OPN on VEGF expression in articular cartilage. Rat articular chondrocytes were isolated and cultured in monolayer conditions, and they were treated with OPN for different time periods (0, 2, 8, 12 or 24 h) and dosages (0, 0.1, 0.25 or 0.5 µM). VEGF expression was assessed by reverse transcription‑quantitative polymerase chain reaction and western blotting. The activation of the phosphoinositide 3‑kinase (PI3K)/AKT and extracellular signal‑regulated kinase (ERK)1/2 pathway was analyzed by detecting the expression of pPI3K, pAKT and pERK1/2. To inhibit the PI3K/AKT and ERK1/2 pathway, LY294002 and PD98059 were used, respectively or in combination. It was identified that OPN increased the expression of VEGF in a dose‑ and time‑dependent manner. The PI3K/AKT and ERK1/2 pathways were activated following OPN stimulation and the effect was concomitant with the upregulation of VEGF. Finally, the regulation of VEGF was inhibited by LY294002 and PD98059, and their combination exhibited a synergistic effect. In conclusion, these findings suggest that OPN may directly upregulate VEGF expression through the PI3K/AKT and ERK1/2 pathway. Further studies are required to reveal the mechanism of action of OPN on cartilage angiogenesis and cartilage destruction.

Aurora-A signaling is activated in advanced stage of squamous cell carcinoma of head and neck cancer and requires osteopontin to stimulate invasive behavior.

The clinical significances, cellular effects, and molecular mechanisms by which Aurora-A mediate its invasive effects in HNSCC are still unclear. Here, we found that Aurora-A expression is significantly higher in tumor tissues on 14-microarray of HNSCC in Oncomine-databases. The activity of Aurora-A was not only found in HNSCC specimens, but also significantly correlated with advanced-T-classification, positive-N-classification, TNM-stage and the poor 5-year survival rate. HNSCC-microarray profile showed that osteopontin and Aurora-A exhibited positive correlation. Stimulation of HNC cells with osteopontin results in an increase in Aurora-A expression where localized at the centrosome. Functionally, Aurora-A had the abilities to stimulate cell motility in HNC cells through increase ERK1/2 activity under osteopontin stimulation. Conversely, depletion of Aurora-A expression by siRNAs suppressed ERK1/2 activity as well as inhibition of cell invasiveness. Treatment with anti-CD44 antibodies in HNC cells not only caused a decrease of mRNA/protein of Aurora-A and ERK1/2 activity upon osteopontin stimulation, but also affected the abilities of Aurora-A-elicited cell motility. Finally, immunohistochemical/Western-blotting analysis of human aggressive HNSCC specimens showed a significant positively correlation between osteopontin-Aurora-A and ERK1/2. These findings suggest that Aurora-A is not only an important prognostic factor but also a new therapeutic target in the osteopontin/CD44/ERK pathway for HNSCC treatment.

Suppression of osteopontin functions by levocetirizine, a histamine H1 receptor antagonist, in vitro.

Objectives. Osteopontin (OPN), a multifunctional glycoprotein secreted from a wide variety of cells after inflammatory stimulation, is well accepted to contribute to the development of allergic diseases. However, the influence of histamine H1 receptor antagonists (antihistamines) on OPN functions is not well understood. The present study was undertaken to examine the influence of antihistamines on OPN functions in vitro. Methods. Human nasal epithelial cells (5 × 105 cells) were stimulated with 250 ng/mL OPN in the presence of either desloratadine (DL), fexofenadine (FEX), or levocetirizine (LCT). The levels of OPN, GM-CSF, Eotaxin, and RANTES in 24 h culture supernatants were examined by ELISA. The influence of LCT on mRNA expression and transcription factor activation in cells were also examined by real-time RT-PCR and ELISA, respectively. Key Findings. The antihistamines examined significantly suppressed the production of GM-CSF, Eotaxin, and RANTES from cells after OPN stimulation. LCT also exhibited the suppression of mRNA expression for chemokines and transcription factor, NF-κB and AP-1, activation, which were increased by the stimulation of cells with OPN. Conclusions. The suppressive activity of LCT on OPN functions on nasal epithelial cells may be responsible for the attenuating effect of the agent on allergic diseases.

Osteopontin increases heme oxygenase–1 expression and subsequently induces cell migration and invasion in glioma cells

Malignant gliomas are associated with high morbidity and mortality because they are highly invasive into surrounding brain tissue, making complete surgical resection impossible. Osteopontin is abundantly expressed in the brain and is involved in cell adhesion, migration, and invasion. The aim of the present study was to investigate the mechanisms of glioma cell migration. Migration and invasion activity were determined by transwell and wound-healing assays. Gene and protein expressions were analyzed by reverse transcription-PCR, real time-PCR, and Western blotting. Nrf2-DNA binding activity was determined by electrophoretic mobility shift assay. Establishment of migration-prone sublines were performed to select highly migratory glioma. An intracranial xenograft mouse model was used for the in vivo study. Application of recombinant human osteopontin enhanced the migration of glioma cells. Expression of heme oxygenase (HO)-1 mRNA and protein also increased in response to osteopontin stimulation. Osteopontin-induced increase in cell migration was antagonized by HO-1 inhibitor or HO-1 small interfering (si)RNA. Osteopontin-mediated HO-1 expression was reduced by treatment with MEK/ERK and phosphatidylinositol 3-kinase/Akt inhibitors, as well as siRNA against Nrf2. Furthermore, osteopontin stimulated Nrf2 accumulation in the nucleus and increased Nrf2-DNA binding activity. In migration-prone sublines, cells with greater migration ability had higher osteopontin and HO-1 expression, and zinc protoporphyrin IX treatment could effectively reduce the enhanced migration ability. In an intracranial xenograft mouse model, transplantation of migration-prone subline cells exhibited higher cell migration than parental tumor cells. These results indicate that osteopontin activates Nrf2 signaling, resulting in enhanced HO-1 expression and cell migration in glioma cells.

FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway

BackgroundNasopharyngeal carcinoma (NPC) is well-known for its highly metastatic characteristics, but little is known of its molecular mechanisms. New biomarkers that predict clinical outcome, in particular the ability of the primary tumor to develop metastatic tumors are urgently needed. The aim of this study is to investigate the role of FLJ10540 in human NPC development.MethodsA bioinformatics approach was used to explore the potentially important regulatory genes involved in the growth/metastasis control of NPC. FLJ10540 was chosen for this study. Two co-expression strategies from NPC microarray were employed to identify the relationship between FLJ10540 and osteopontin. Quantitative-RT-PCR, immunoblotting, and immunohistochemistry analysis were used to investigate the mRNA and protein expression profiles of FLJ10540 and osteopontin in the normal and NPC tissues to confirm microarray results. TW01 and Hone1 NPC cells with overexpression FLJ10540 or siRNA to repress endogenous FLJ10540 were generated by stable transfection to further elucidate the molecular mechanisms of FLJ10540-elicited cell growth and metastasis under osteopontin stimulation.ResultsWe found that osteopontin expression exhibited a positive correlation with FLJ10540 in NPC microarray. We also demonstrated comprehensively that FLJ10540 and osteopontin were not only overexpressed in NPC specimens, but also significantly correlated with advanced tumor and lymph node-metastasis stages, and had a poor 5-year survival rate, respectively. Stimulation of NPC parental cells with osteopontin results in an increase in FLJ10540 mRNA and protein expressions. Functionally, FLJ10540 transfectant alone, or stimulated with osteopontin, exhibited fast growth and increased metastasis as compared to vehicle control with or without osteopontin stimulation. Conversely, knockdown of FLJ10540 by siRNA results in the suppression of NPC cell growth and motility. Treatment with anti-CD44 antibodies in NPC parental cells not only resulted in a decrease of FLJ10540 protein, but also affected the abilities of FLJ10540-elicited cell growth and motility in osteopontin stimulated-NPC cells.ConclusionsThese findings suggest that FLJ10540 may be critical regulator of disease progression in NPC, and the underlying mechanism may involve in the osteopontin/CD44 pathway.

Osteopontin induces β-catenin signaling through activation of Akt in prostate cancer cells

Secretion of osteopontin (OPN) by cancer cells is a known mediator of tumorigenesis and cancer progression in both experimental and clinical studies. Our work demonstrates that OPN can activate Akt, an important step in cancer progression. Both ILK and PI3K are integral proteins in the OPN/Akt pathway, as inhibition of either kinase leads to a loss of OPN-mediated Akt activation. Subsequent to OPN-induced Akt activation, we observe inactivation of GSK-3β, a regulator of β-catenin. Osteopontin stimulation leads to an overall increase in β-catenin protein levels with a resultant transfer of β-catenin to the nucleus. Through the nuclear import of β-catenin, OPN increases both the transcription and protein levels of MMP-7 and CD44, which are known TCF/LEF transcription targets. This work describes an important aspect of cancer progression induced by OPN.

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin, FAK, ERK, and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, alphavbeta3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, alphavbeta3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN-induced increase of the migration and MMP-9 up-regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, treatment of JJ012 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced cell migration and MMP-9 up-regulation. Stimulation of JJ012 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in MMP-9 and kappaB-luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the alphavbeta3 integrin, FAK, MEK, ERK and NF-kappaB signal transduction pathway.

Osteopontin stimulates invasion of NCI‐h295 cells but is not associated with survival in adrenocortical carcinoma

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin alphavbeta3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin alphavbeta3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin alphavbeta3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin beta3 antibody, indicating a functional relationship of OPN and integrin alphavbeta3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin alphavbeta3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.

Osteopontin increases lung cancer cells migration via activation of the αvβ3 integrin/FAK/Akt and NF-κB-dependent pathway

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which is abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and cell proliferation via interaction with its receptor, αvβ3 integrin. However, the effect of OPN on migration activity in human lung cancer cells is mostly unknown. Here we found that OPN increased the migration via activation of αvβ3 integrin in human lung cancer cells (A549 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002), Akt inhibitor or ERK inhibitor (PD98059) inhibited the OPN-induced increase in the migration of lung cancer cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), p85 subunit of PI3K, serine 473 of Akt and ERK. In addition, treatment of A549 cells with NF-κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN-induced migration of lung cancer cells. Stimulation of A549 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser 536 phosphorylation, and κB-luciferase activity. The OPN-mediated increases in IKK α/β, IκBα and p65 Ser 536 phosphorylation were inhibited by Ly294002, Akt inhibitor and PD98059. Co-transfection with FAK, p85, Akt and ERK mutants also reduced the OPN-induced κB-luciferase activity. Taken together, these results suggest that OPN acts through αvβ3 integrin, which in turn activates the FAK, PI3K, Akt, ERK and NF-κB pathways, contributing to the migration of lung cancer cells.

Osteopontin, a Protein with Cytokine‐like Properties, is Associated with Inflammation in Crohn's Disease

In Crohn's disease (CD) mucosal T-cells produce increased interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) levels and TNF-alpha antibody treatment [Infliximab (Ifx)] is effective. Osteopontin (OPN), a glycoprotein stimulating activated T-lymphocytes, may be involved in the disturbed immune-regulation but also in normal immune-homeostasis and mucosal repair, since it is expressed in many tissues and present in human milk. This study investigates plasma-OPN levels in CD patients during Ifx treatment and the in vitro effect of OPN on intestinal T cells. Thirty-seven CD patients received three Ifx doses at week 0, 2 and 6. Blood samples, colonic biopsies and clinical scores were obtained before treatment and at week 8, 26 and 52. In-vivo activated T-cell cultures were established from colonic biopsies in the presence of interleukin (IL)-2 and IL-4. The in vitro effect of OPN stimulation on T-cell IFN-gamma, TNF-alpha, and IL-10 production was measured. Plasma-OPN was increased in active CD (increased CRP-level) compared with quiescent disease (P = 0.02) and declined after three Ifx doses (P = 0.04). It was inversely correlated with in vitro T-cell IL-10 production. OPN increased CD69 and CD25 expression and enhanced T-cell IFN-gamma and TNF-alpha production in a dose-dependent fashion with higher levels in CD than in healthy controls (HC), but induced a concomitant higher IL-10 production in HC than CD. In conclusion, plasma-OPN levels are related to CD inflammation. In vitro, OPN-stimulated IL-10 production increases less in T-cell cultures from CD patients than from HC, indicating that IL-10 deficiency may be involved in the defect immune-regulation in CD, even after OPN stimulation.

Phosphorylation of a Wiscott-Aldrich Syndrome Protein-associated Signal Complex Is Critical in Osteoclast Bone Resorption

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.

Angiogenesis and Osteoclastogenesis Are Mutually Stimulated in Myeloma: A Role for VEGF and Osteopontin.

Angiogenesis and osteoclastogenesis occur in the bone marrow as myeloma (MM) progresses. Suppression of osteoclastogenesis by bisphosphonates reduced tumor burden as well as vascular density in the BM in myeloma animal models(Croucher et al. 2003). These evidences suggest a link between angiogenesis, osteoclastogenesis and MM tumor expansion which orchestrates a network of co-operative cellular interactions. We have previously demonstrated that a cell-cell interaction between osteoclasts (OCs) and MM cells potently enhanced MM cell growth and survival as well as angiogenesis. Such angiogenic effects were almost completely abrogated by antibodies against VEGF and osteopontin (OPN) in combination, suggesting VEGF and OPN as predominant angiogenic factors produced in MM bone marrow microenvironment. Angiogenesis is a multi-step process including migration, proliferation and sprouting in response to angiogenic stimuli. The impact of these factors on these angiogenic processes as well as on osteoclastogenesis by vascular cells is largely unknown. We therefore investigated roles for OPN and VEGF in combination in different steps in angiogenesis and in induction of osteoclastogenic activity by vascular endothelial cells. Conditioned media (CM) from co-cultures of peripheral blood mononuclear cell-derived OCs and RPMI8226 cells at 10 % enhanced survival of HUVEC under factor-deprived conditions 3-fold compared to control culture media, and transmigration of HUVEC 1.5-fold. Antibodies against VEGF and OPN each alone partially and both in combination mostly abrogated these effects of the CM. Consistently, VEGF and OPN co-operatively promoted survival and transmigration of HUVEC as well as angiogenesis in vitro. In order to further clarify a role for VEGF and OPN in a link between angiogenesis and osteoclastogenesis, we next examined osteoclastogenic activity produced by HUVEC upon VEGF and OPN stimulation using rabbit bone cell cultures on dentine slices. CM were collected from HUVEC cultured on either OPN- or type 1 collagen-coated wells in the presence or absence of VEGF. CM from OPN-coated wells with VEGF enhanced OC formation most potently(2.8-fold) compared to control culture media, while CM from OPN-coated wells without VEGF, type 1 collagen-coated wells with VEGF and type 1 collagen-coated wells without VEGF enhanced 2-, 1.8- and 1.2-fold, respectively, indicating co-operative actions of VEGF and OPN. Furthermore, such VEGF and OPN treatment was found to induce osteoclastogenic IL-8 by HUVEC most apparently among 125 different cytokines determined by cytokine antibody arrays. These results suggest that VEGF and OPN derived from MM cells and OCs cause angiogenesis as well as osteoclastogenesis at least in part through the enhancement of migration and survival and the induction of osteoclastogenic activity of vascular endothelial cells, thereby mutually stimulating angiogenesis and osteoclastogenesis. Therefore, VEGF and OPN may be a therapeutic target against MM bone marrow environment to ameliorate bone destruction along with MM expansion.

Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells.

The interaction of osteopontin (OPN) with CD44 and alphavbeta3-integrin has been implicated in numerous signal transduction pathways that may promote cancer metastasis. CD44v6 is a splice variant of CD44 which has been identified as a marker of cancer progression. In this study, immortalized liver carcinoma cells (HepG2) were used to examine the effect of OPN on two isoforms of CD44: CD44 standard (CD44 s) and CD44v6. Western blots demonstrated that OPN up-regulated plasma membrane CD44v6 protein expression in a concentration- and time-dependent fashion. CD44v6 levels returned to control levels when OPN-alphavbeta3-integrin binding was blocked by an RGD peptide or tyrosine kinase activity was inhibited. OPN significantly increased CD44v6 protein synthesis, while simultaneously decreasing protein degradation. Steady-state mRNA levels of both CD44s and CD44v6 were unaltered in the presence of OPN stimulation. OPN increased HepG2 in vitro adhesion to hyaluronate (HA); excess soluble HA extinguished OPN-mediated HepG2 adhesion, indicating CD44 dependence. In conclusion, OPN binds to the alphavbeta3-integrin to increase plasma membrane CD44v6 expression and augment in vitro adhesion to HA. This may contribute to the mechanism by which OPN enhances metastatic behavior in hepatocellular cancer cells.

Osteopontin inhibits expression of cytochrome c oxidase in RAW 264.7 murine macrophages

Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two CAM molecules: α vβ 3-integrin and CD44. OPN initiates a number of signal transduction pathways that control cell survival, proliferation, and migration. In this study, utilizing RAW 264.7 murine macrophages, we demonstrate that expression of the mitochondrial protein, CCOI, is significantly decreased in the setting of OPN stimulation. This effect is blocked by the CD44 competitive ligand, hylauronate; GRGDSP, a hexapeptide that blocks OPN–integrin binding, had no effect. CCOI mRNA and transcription were significantly decreased in the presence of OPN; CCOI mRNA half-life was unaltered by OPN. Additional mitochondrial run-on studies, which included genes from L-strand and H-strand, suggest that OPN terminates transcription of the distal H-strand. CCO enzyme activity was also significantly decreased by OPN. Our results indicate that OPN inhibits CCOI expression as the result of a novel CD44-dependent transcriptional regulatory mechanism of the mitochondrial H strand.

The integrin alpha(v)beta(3) and CD44 regulate the actions of osteopontin on osteoclast motility.

In the studies reported here we demonstrate that osteopontin is secreted from the basolateral surfaces of osteoclasts where it binds to the avb3-integrin, suggesting that it may be an autocrine factor. Osteopontin stimulation of osteoclasts produced changes in cell shape by causing disruption of peripheral podosome structures and formation of actin filaments at the leading edge of the migrating osteoclasts. The latter was part of the assumption of a motile phenotype prior to cells reforming peripheral ring type podosome containing clear zones. It is well established in our laboratory as well as in others that osteopontin stimulated osteoclast motility and bone resorption. The effect of osteopontin was mimicked by RGD containing peptides and blocked by a avb3 antibody, demonstrating that signals generated by integrin ligation contributed to the actions of osteopontin. In addition, the migratory effects of osteopontin on osteoclasts were also mediated through CD44 receptors since blocking antibodies to CD44 blocked stimulation of motility. Our data strongly suggest that osteopontin is an osteoclast autocrine motility factor binding to avb3 and CD44 during stimulation of osteoclast migration.

Osteopontin augments CD3-mediated interferon-γ and CD40 ligand expression by T cells, which results in IL-12 production from peripheral blood mononuclear cells

Osteopontin is an RGD-containing bone matrix protein with cytokine-like functions that is associated with early stages of Th1-mediated diseases. Although the function of osteopontin in these responses is unknown, it is expressed by activated T cells and macrophages and can costimulate T cell proliferation. Studies have demonstrated that early IL-12 and IFN-gamma expression is required to induce a protective response to many intracellular pathogens. Herein, we demonstrate that osteopontin stimulation augments the ability of anti-CD3 monoclonal antibody to induce CD40 ligand (CD40L) and IFN-gamma expression on human T cells, resulting in CD40L- and IFN-gamma-dependent IL-12 production in vitro. These findings suggest a functional role for osteopontin in early Th1 responses, namely regulation of T cell-dependent IL-12 production. Further, osteopontin up-regulation of CD40L provides mechanistic support for the association of osteopontin with polyclonal B cell proliferation and humoral autoimmune disease.

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts.

Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.