Functional Characterization of Stromal Osteopontin in Melanoma Progression and Metastasis

Background

Recent studies demonstrated that not only tumor derived- but stroma derived factors play crucial role in cancer development. Osteopontin (OPN) is a secreted non-collagenous, sialic acid rich, chemokine-like phosphoglycoprotein that facilitates cell-matrix interactions and promotes tumor progression. Elevated level of OPN has been shown in melanoma patient and predicted as a prognostic marker. Recent reports have indicated that stroma-derived OPN are involved in regulating stem cell microenvironment and pre-neoplastic cell growth. However, the function of stroma derived OPN in regulation of side population (SP) enrichment leading to melanoma growth, angiogenesis and metastasis is not well studied and yet to be the focus of intense investigation.

Methodology/Principal Findings

In this study, using melanoma model, in wild type and OPN knockout mice, we have demonstrated that absence of host OPN effectively curbs melanoma growth, angiogenesis and metastasis. Melanoma cells isolated from tumor of OPN wild type (OPN^+/+) mice exhibited more tumorigenic feature as compared to the parental cell line or cells isolated from the tumors of OPN KO (OPN^−/−) mice. Furthermore, host OPN induces VEGF, ABCG2 and ERK1/2 expression and activation in B16-WT cells. We report for the first time that stroma derived OPN regulates SP phenotype in murine melanoma cells. Moreover, loss in and gain of function studies demonstrated that stroma-derived OPN regulates SP phenotype specifically through ERK2 activation.

Conclusions

This study establish at least in part, the molecular mechanism underlying the role of host OPN in melanoma growth and angiogenesis, and better understanding of host OPN-tumor interaction may assist the advancement of novel therapeutic strategy for the management of malignant melanoma.     

Establishment and characterization of an osteopontin-null cutaneous squamous cell carcinoma cell line

Osteopontin (OPN) is a secreted glycoprotein implicated to function in cancer development and metastasis. Although elevated expression of OPN are observed in cancer cells of various types, in some cases, only the cells in the stromal region surrounding the tumor express OPN, suggesting distinct functional roles for this protein derived from host cells and from cancer cells. To provide a model for addressing the functions and mechanisms of host-derived OPN in cancer progression and metastasis, a cutaneous squamous cell carcinoma cell line (ONSC) that lacks the OPN gene, Spp1, was established. This line of cells was derived from a squamous cell carcinoma that developed in a female, OPN-null mouse subjected to two-stage skin carcinogenesis. Morphologically, ONSC cells resemble epithelial cells, and they express the epithelial markers, K1, K14, and p63, as confirmed by immunohistochemical analyses. Genomic analyses indicate the presence of mutated H-Ras and p53 genes. ONSC cells form colonies in soft agar and, subcutaneously injected into athymic nude mice, develop into squamous cell carcinomas that metastasize to the lungs. Lacking OPN expression, these squamous cell carcinoma cells provide a model to address the function of host OPN in the context of cancer progression and metastasis.     

Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Osteopontin in metastatic lesions as a prognostic marker in ovarian cancers

Summary Osteopontin (OPN) is expressed in various human cancers and associated with tumor progression, invasion and metastasis in many manners. The purpose of this study is to investigate the clinical significance of OPN expression in metastatic lesions of ovarian cancers, since the prognosis of the patients with peritoneal dissemination is extremely poor. In primary tumors and peritoneal metastatic lesions from 40 patients with stage III ovarian cancers, the protein levels of OPN and histoscores were determined by enzyme immunoassay and immunohistochemistry, respectively. Immunohistochemical staining revealed OPN was distributed in the cytoplasm and nuclear compartments of the cancer and stromal cells within and around the tumor. The OPN level was significantly (p < 0.05) increased in 32 of 40 metastatic lesions of ovarian cancers. The OPN increased cases identified by immunohistochemical staining were consistent with those identified by the sandwich immunoassay. The prognosis of the 32 patients with significant increase of OPN in ovarian cancers was extremely poor, whereas the 36-month survival rate of the 8 patients with no increase of OPN was 75%. Multivariate analysis revealed that the levels of OPN were independent predictors of prognosis from clinical characteristics (age, lesion size, histological types). OPN might be associated with peritoneal metastasis and its advancement, and that the OPN level in metastatic lesion may be a prognostic indicator in ovarian cancers.     

The RGD Domain of Human Osteopontin Promotes Tumor Growth and Metastasis through Activation of Survival Pathways

Background

Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models.

Methodology/Principal Findings

Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-κB activation and FAK phosphorylation. Inhibition of NF-κB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells.

Conclusion/Significance

Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-κB activation.     

The inhibition of in vivo tumorigenesis of osteosarcoma (OS)-732 cells by antisense human osteopontin RNA

Osteopontin (OPN) is a secreted, non-collagenous, sialic acid-rich protein which functions by mediating cell-matrix interactions and cellular signaling via binding with integrins and CD44 receptors. An increasing number of studies have shown that OPN plays an important role in controlling cancer progression and metastasis. OPN was found to be expressed in many human cancer types, and in some cases, its over-expression was shown to be directly associated with poor patient prognosis. In vitro cancer cell line and animal model studies have clearly indicated that OPN can function in regulating the cell signaling that ultimately controls the oncogenic potential of various cancers. Previous studies in our laboratory demonstrated that OPN is highly expressed in human osteosarcoma (OS) cell line OS-732. In this study, we successfully reduced the tumorigenecity of OS-732 cells xenotransplanted into nude mice, using the antisense human OPN (hOPN) RNA expression vector.     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC₅₀ value of 46 ± 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [³H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

In vitro and clinical data analysis of Osteopontin as a prognostic indicator in colorectal cancer

Osteopontin (OPN) has been shown to promote colorectal cancer (CRC) progression; however, the mechanism of OPN‐induced CRC progression is largely unknown. In this study, we found that OPN overexpression led to enhanced anchorage‐independent growth, cell migration and invasion in KRAS gene mutant cells but to a lesser extent in KRAS wild‐type cells. OPN overexpression also induced PI3K signalling, expression of Snail and Matrix metallopeptidase 9 (MMP9), and suppressed the expression of E‐cadherin in KRAS mutant cells. In human CRC specimens, a high‐level expression of OPN significantly predicted poorer survival in CRC patients and OPN expression was positively correlated with MMP9 expression, and negatively correlated with E‐cadherin expression. Furthermore, we have found that 15 genes were co‐upregulated in OPN highly expression CRC and a list of candidate drugs that may have potential to reverse the secreted phosphoprotein 1 (SPP1) gene signature by connectivity mapping. In summary, OPN is a potential prognostic indicator and therapeutic target for colon cancer.     

Overexpression of osteopontin in hepatocellular carcinoma and its relationships with metastasis,invasion of tumor cells

Osteopontin (OPN) plays an important role in metastasis and relapse of human cancer. However, the whole story of OPN relating to cancer has been far from clear untill now. To investigate the expression of OPN in hepatocellular carcinoma (HCC) and its relationships with recurrence and metastasis of HCC, normal and malignant liver tissues from patients with HCC were analyzed using immunohistochemical staining. OPN expression was inhibited by small interfering RNA (siRNA) in HCC cells lines, and then colony formation and matrigel invasion were examined. The results showed that expression of OPN was associated with metastasis of HCC with a positive rate of OPN in the tissue of HCC (70.00%), which was highly more obvious than those in paracarcinoma tissue and normal liver tissue (P < 0.01). In HCC cell lines, OPN depletion could reduce formed colony and metastasizing numbers in vitro. In conclusion, Expression of OPN in the tissue of HCC is related to metastasis or metastases. Specific siRNA could decrease expressions of OPN at both mRNA and protein levels, and abates the invasiveness of hepatocellular carcinoma cells, suggesting that OPN might be a promising agent for treatment of metastasis and recurrence of HCC.     

FGF‐8b induces growth and rich vascularization in an orthotopic PC‐3 model of prostate cancer

Fibroblast growth factor 8 (FGF‐8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF‐8b on proliferation of PC‐3 prostate cancer cells and growth of PC‐3 tumors, and to identify FGF‐8b‐associated molecular targets. Expression of ectopic FGF‐8b in PC‐3 cells caused a 1.5‐fold increase in cell proliferation in vitro and a four‐ to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF‐8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT‐qPCR of FGF‐8b mRNA. Microarray analyses revealed significantly altered, up‐ and downregulated, genes in PC‐3 cell cultures (169 genes) and in orthotopic PC‐3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell‐to‐cell signaling and energy production, and the downregulated genes associated with differentiation (DKK‐1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT‐qPCR. In conclusion, our results demonstrate that FGF‐8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF‐8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. 107: 769–784, 2009. © 2009 Wiley‐Liss, Inc.     

Osteopontin promotes hepatocellular carcinoma invasion by up-regulating MMP-2 and uPA expression

Osteopontin (OPN) is over-expressed in a variety of cancers, but its role in hepatocellular carcinoma (HCC) progression has not been clarified. In this study, weakly tumorigenic, non-metastastic human HCC cell line SMMC-7721 cells were forced to over-express OPN via stable transfection. A series of functional assays were performed to assess the effects of OPN on tumor cell behaviors and cDNA microarray was used to identify the genes regulated by OPN. The results showed that OPN significantly enhanced the migration and invasion of SMMC-7721 cells in vitro. In addition, CD44v6 antibody could significantly inhibit the invasion of OPN over-expressing SMMC-7721 cells. Moreover, MMP-2 and uPA expressions were significantly up-regulated in OPN over-expressing SMMC-7721 cells. Together, these findings indicate that OPN enhanced HCC cells invasion through interaction with its receptor CD44v6 and increased MMP-2 and uPA expressions, providing at least one mechanism for OPN-mediated HCC progression and metastasis.     

Extensive Clear Zone and Defective Ruffled Border Formation in Osteoclasts of Osteopetrotic (ia/ia) Rats: Implications for Secretory Function

The cellular distribution of osteoclast integrin subunits α_v and β₃, the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits α_v and β₃ were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane.In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.     

The functional and clinical roles of osteopontin in cancer and metastasis

Osteopontin (OPN) is a secreted and integrin-binding protein that has been implicated in a number of pathologies. In this review we will focus on the functional and clinical roles of OPN in cancer and metastasis, with a particular emphasis on breast cancer. While much evidence has suggested that OPN is associated with cancer, its functional contribution to cancer remains poorly understood. Here we will review evidence for mechanisms by which OPN may act to enhance malignancy, including evidence that signaling pathways directly induced by OPN, as well as interactions with growth factor receptor pathways, can combine to activate expression of genes and functions that contribute to metastasis. OPN has been shown to be over-expressed in a variety of human tumors and is present in elevated levels in the blood of some patients with metastatic cancers. We also will discuss recent clinical evidence that suggests that OPN is not only associated with several tumor types, but that levels of OPN in cancer patients' blood or tumors may provide prognostic information.     

Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma In Vivo

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.     

术前CA125、OPN、CXCL8、NLR联合检测对乳腺癌改良根治术患者术后复发转移风险的评估价值

摘要 目的：探讨术前糖类抗原125（CA125）、骨桥蛋白（OPN）、趋化因子配体8（CXCL8）、中性粒细胞与淋巴细胞比值（NLR）联合检测对乳腺癌改良根治术患者术后复发转移风险的评估价值。方法：选取2015年4月-2016年4月期间我院收治的乳腺癌改良根治术患者384例按照术后有无复发转移分为未复发转移组（n=345）和复发转移组（n=39）,对比复发转移组、未复发转移组CA125、OPN、CXCL8、NLR,乳腺癌改良根治术患者术后复发转移的影响因素采用多因素Logistic回归分析。采用受试者工作特征（ROC）曲线来判断CA125、OPN、CXCL8、NLR检测对乳腺癌改良根治术患者术后复发转移风险的评估价值。结果：复发转移组的CA125、OPN、CXCL8、NLR高于未复发转移组,组间对比差异有统计学意义（P<0.05）。乳腺癌改良根治术患者术后复发转移与肿瘤最大直径、临床分期、术前新辅助化疗、人表皮生长因子受体2（HER2）、淋巴结转移、组织学类型、细胞增殖标志抗原（ki-67）、雌激素受体（ER）/孕激素受体（PR）、P53、术后放疗、术后内分泌治疗有关（P<0.05）。多因素Logistic回归分析结果显示：OPN偏高、CXCL8偏高、NLR偏高、肿瘤最大直径≥2 cm、淋巴结转移阳性、ER/PR双阴性、临床分期为III期、术前未接受新辅助化疗是乳腺癌改良根治术患者术后复发转移的危险因素（P<0.05）。术前CA125、OPN、CXCL8、NLR联合检测评估复发转移的曲线下面积（AUC）为0.855均高于各指标单独检测。结论：乳腺癌改良根治术后复发转移与OPN、CXCL8、NLR、肿瘤最大直径、淋巴结转移、ER/PR、临床分期、术前接受新辅助化疗均存在一定联系,临床需据此采取针对性干预措施加以防范。且术前CA125、OPN、CXCL8、NLR联合检测辅助评估术后复发转移的价值较高。     

Cks1 regulates human hepatocellular carcinoma cell progression through osteopontin expression

Precise cell cycle regulation is critical to prevent aberrant cell proliferation and cancer progression. Cks1 was reported to be an essential accessory factor for SCF^Skp2, the ubiquitin ligase that targets p27^Kip1 for proteasomal degradation; these actions drive mammalian cell transition from G1 to S phase. In this study, we investigated the role played by Cks1 in the growth and progression of human hepatocellular carcinoma (HCC) cells. Silencing Cks1 expression abrogated osteopontin (OPN) expression in a p27^Kip1-dependent manner in Huh7 HCC cells. OPN increased the proliferation, migration and invasion of Huh7 cells. Pharmacological inhibitor studies demonstrated that ERK1/2 signaling is responsible mainly for Cks1-mediated OPN expression. Cks1 appears to regulate ERK1/2 signaling through the expression of dual-specificity phosphatase 16 (DUSP16) because both Cks1 knockdown, which leads to DUSP16 upregulation, and DUSP16 overexpression decreased ERK1/2 phosphorylation and the resulting OPN expression. The same is true for the Cks1-mediated increases in p27^Kip1, suggesting that Cks1 regulates OPN expression through activating ERK1/2 signaling either by suppressing DUSP16 expression or by a p27^Kip1-dependent mechanism. Cks1 and OPN expression levels were significantly higher, but DUSP16 expression levels were significantly lower in HCC tissues than in normal liver tissues. Both Cks1 and OPN expression were negatively correlated with DUSP16 expression, whereas Cks1 expression was positively correlated with OPN expression. Moreover, combined panels for the expression levels of Cks1, DUSP16 and OPN showed significant prognostic power for the risk assessment of HCC patient overall survival. In conclusion, our data propose a novel function for Cks1 as a tumor promoter through the expression of the strongly oncogenic protein OPN in HCC.     

Genetic variations in the SPP1 promoter affect gene expression and the level of osteopontin secretion into bovine milk

Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin‐binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.     

Osteopontin mediates macrophage chemotaxis via α4 and α9 integrins and survival via the α4 integrin

Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to α_v‐containing integrins, and the SLAYGLR sequence binding to α₄β₁, α₄β₇, and α₉β₁ integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated‐bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α₄ integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α₄ and α₉ integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL‐12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid‐derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate‐elicited peritonitis. Collectively, these data indicate that α₄ and α₉ integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013. © 2012 Wiley Periodicals, Inc.