Wild-type Osteopontin Research Articles

Mechanistic Insights into the Role of OPN in Mediating Brain Damage via Triggering Lysosomal Damage in Microglia/Macrophage.

We previously found that osteopontin (OPN) played a role in hypoxia-ischemia (HI) brain damage. However, its underlying mechanism is still unknown. Bioinformatics analysis revealed that the OPN protein was linked to the lysosomal cathepsin B (CTSB) and galectin-3 (GAL-3) proteins after HI exposure. In the present study, we tested the hypothesis that OPN was able to play a critical role in the lysosomal damage of microglia/macrophages following HI insult in neonatal mice. The results showed that OPN expression was enhanced, especially in microglia/macrophages, and colocalized with lysosomal-associated membrane protein 1 (LAMP1) and GAL-3; this was accompanied by increased LAMP1 and GAL-3 expression, CTSB leakage, as well as impairment of autophagic flux in the early stage of the HI process. In addition, the knockdown of OPN expression markedly restored lysosomal function with significant improvements in the autophagic flux after HI insult. Interestingly, cleavage of OPN was observed in the ipsilateral cortex following HI. The wild-type OPN and C-terminal OPN (Leu152-Asn294), rather than N-terminal OPN (Met1-Gly151), interacted with GAL-3 to induce lysosomal damage. Furthermore, the secreted OPN stimulated lysosomal damage by binding to CD44 in microglia in vitro. Collectively, this study demonstrated that upregulated OPN in microglia/macrophages and its cleavage product was able to interact with GAL-3, and secreted OPN combined with CD44, leading to lysosomal damage and exacerbating autophagosome accumulation after HI exposure.

Osteopontin Undergoes Polymerization in Vivo and Gains Chemotactic Activity for Neutrophils Mediated by Integrin α9β1

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.

Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53

Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53(-/-) mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.

Osteopontin Overexpression Inhibits in Vitro Re-endothelialization via Integrin Engagement

The extracellular matrix protein osteopontin (OPN) plays a nonredundant role in atherosclerosis and restenosis. Here we investigated the impact of OPN up-regulation in an in vitro model of re-endothelialization after mechanical injury of the endothelial cell monolayer. Murine aortic endothelial (MAE) cells interact via alpha(v) integrins with the integrin-binding Arg-Gly-Asp OPN sequence and adhere to immobilized OPN. On this basis, MAE cells were stably transfected with a wild-type OPN cDNA (OPN-MAE cells), with an OPN mutant lacking the Arg-Gly-Asp sequence (DeltaRGD-OPN-MAE cells), or with vector alone (mock-MAE cells). When compared with mock-MAE and DeltaRGD-OPN-MAE cells, OPN-MAE cells showed a reduced sprouting activity in fibrin gel, a reduced motility in a Boyden chamber assay, and a reduced capacity to repair the wounded monolayer. Accordingly, OPN-MAE cells at the edge of the wound were unable to form membrane ruffles, to reorganize their cytoskeleton, and to activate the focal adhesion kinase and the small GTPase Rac1, key regulators of the cell entry into the first phase of the cell migration cycle. Accordingly, wounded OPN-MAE cells failed to activate the intracellular signals RhoA and ERK1/2, involved in the later phases of the cell migration cycle. Also, parental MAE cells showed reduced re-endothelialization after wounding when seeded on immobilized OPN and exhibited increased adhesiveness to OPN-enriched extracellular matrix. In conclusion, OPN up-regulation impairs re-endothelialization by inhibiting the first phase of the cell migration cycle via alpha(v) integrin engagement by the extracellular matrix-immobilized protein. This may contribute to the adverse effects exerted by OPN in restenosis and atherosclerosis.

Generation of a BCR-ABL-Induced Leukemia Model in Osteopontin-Deficient Mice.

Osteopontin (OPN) is a secreted phoshoprotein playing multiple roles in cell migration, apoptosis and angiogenesis with evidence of increased expression in several types of epithelial tumors. Recently, OPN has also been shown to be a key regulator of the migration of hematopoietic stem cells towards the osteoblastic niche where it participates to the maintenance of the quiescence of stem cells after binding to integrins. We have previously shown that OPN is upregulated by BCR-ABL in a tyrosine-kinase-dependent manner in a TET-regulated BCR-ABL expressing cell line. To further study the potential role of OPN in BCR-ABL-associated leukemogenesis, we have used a retrovirus mediated BCR-ABL gene transfer strategy in OPN-deficient mice (OPN−/−). Bone marrow stem cells were harvested from OPN−/− and wild type OPN+/+ (C57Bl/6) mice after 5-FU treatment at day-5 and retrovirally transduced by a MIGR-p210 BCR-ABL vector in the presence of cytokines for 3 days. Retrovirally transduced marrow cells were transplanted into lethally irradiated OPN−/− mice in the presence of appropriate irradiation controls. Transplanted animals were followed regularly by blood counts, appearance of clinical disease and the results were compared to those obtained in OPN+/+ mice transplanted in the same conditions. In preliminary experiments, we have established that BCR-ABL-transduced OPN+/+ marrow can generate leukaemia in the background of OPN−/− mice with similar efficiencies (n=3) as in the background of OPN+/+ mice (n=3). In following experiments we have observed excessive mortality after irradiation (9–9.5 Gys) and established the optimal protocol pour induction of leukaemia in OPN−/− mice transplanted with OPN−/− cells. We have found that co-transplantation of 5.104 retrovirally transduced BM cells in the presence of 1.5 105 untransduced bone marrow allows reproducibly leukemia induction. The majority of OPN−/− mice (20/24, n=2 experiments) transplanted by the use of this protocol developed hyperleucocytosis between day 21–35 after transplantation. The latency of leukaemia did not differ between wild type and OPN−/− animals. At the time of sacrifice, all mice were found to have splenomegaly (500–950 mg). Both marrow and spleen cells had massive infiltration with GFP+ cells (40–80 %, marrow; 40–90%, spleen). At phenotypic analysis, OPN−/− leukemic cells were found to express at variable degrees Gr1, CD11b, Ter119, CD19, suggesting the establishment of a multilineage disease in the background of OPN−/− marrow. The phenotypic analysis leukemic cells from OPN+/+ mice was found to be similar. Culture of spleen cells from of a leukemic OPN−/− mouse gave rise to a GFP+ cell line expressing c-kit (97%) with negativity of Gr1, B220, Ter119 expression, suggesting its mast cell phenotype. Interestingly, this cell line has exquisite sensitivity to imatinib mesylate, the cytotoxicity of which is reduced by IL-3 but not by stem cell factor. In summary, these results demonstrate that OPN, a transcriptional target of BCR-ABL oncogene, is not required for BCR-ABL-induced murine leukemogenesis. It remains to be determined if it plays a role in the stem cell quiescence of BCR-ABL-expressing primitive cells in the osteoblastic niche and their resistance to BCR-ABL-targeted therapies. The OPN−/− murine CML modelthat we have established will be used to explore these questions as well as the homing potential of leukemic cells in the presence or in the absence of OPN.

Role of the Integrin-Binding Protein Osteopontin in Lymphatic Metastasis of Breast Cancer

Although a primary route of breast cancer metastasis is believed to be via lymphatics, the molecular factors involved are poorly understood. We hypothesized that one such factor may be the integrin-binding protein osteopontin (OPN), and we investigated this clinically and experimentally. In breast cancer patients undergoing sentinel lymph node biopsy, OPN levels were significantly higher in lymph node metastases than in the primary tumor (P < 0.001). To test the functional contribution of OPN to lymphatic metastasis and to determine whether the RGD (Arg-Gly-Asp) integrin-binding sequence of OPN is important for this process, we transfected wild-type OPN or mutant OPN (lacking the RGD sequence) into MDA-MB-468 human breast cancer cells. In vitro, cells overexpressing OPN demonstrated increased anchorage-independent growth in soft agar (P = 0.001) and increased RGD-dependent adhesion (P = 0.045). Following mammary fat pad injection of nude mice, cells overexpressing OPN showed increased lymphovascular invasion, lymph node metastases, and lung micrometastases at earlier time points (P = 0.024). Loss of the RGD region partially abrogated this effect in the lymphatics (P = 0.038). These novel findings indicate that OPN is a key molecular player involved in lymphatic metastasis of breast cancer, potentially by affecting RGD-mediated adhesive interactions and by enhancing the establishment/persistence of tumor cells in the lymphatics.

S-Nitrosylation of Heterogeneous Nuclear Ribonucleoprotein A/B Regulates Osteopontin Transcription in Endotoxin-stimulated Murine Macrophages

Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids that contains a Gly-Arg-Gly-Asp-Ser sequence. It is a secreted protein with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. Despite the many recognized functions of OPN, very little is known of the transcriptional regulation of OPN. In this regard, we have previously demonstrated that OPN transcription and promoter activity are significantly up-regulated in response to NO in a system of endotoxin-stimulated murine macrophages. However, the specific cis- and trans-regulatory elements that determine the extent of endotoxin- and NO-mediated induction of OPN synthesis are unknown. In this follow-up study, we demonstrate that: 1) OPN gene transcription is regulated by a constitutive transcriptional repressor protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B); 2) inhibition of in vivo hnRNP DNA binding activity is accompanied by increased S-nitrosylation of hnRNP A/B in the setting of lipopolysaccharide (LPS)-mediated NO synthesis; 3) inhibition of LPS mediated NO synthesis restores hnRNP DNA binding and decreases the extent of S-nitrosylation; and 4) S-nitrosylation of hnRNP at cysteine 104 inhibits in vitro DNA binding activity, which is reversed by dithiothreitol. Our findings suggest that LPS induced S-nitrosylation of hnRNP inhibits its activity as a constitutive repressor of the OPN promoter and results in enhanced OPN expression.

The Osteopontin-CD44 Survival Signal Involves Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colony-stimulating factor signaling pathways and that, through binding to the cell surface receptor CD44, OPN contributes to the survival activities of interleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S.-T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000) Mol. Cell. Biol. 20, 2734-2742). In this report, we demonstrate that the CD44-binding domain of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of either residue with alanine results into a dominant negative mutant that overrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidylinositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt activation are detected in IL-3-treated cells and that the survival promoting effect of OPN is mediated predominantly through the phosphatidylinositol 3-kinase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.

Identification of Dual α4β1 Integrin Binding Sites within a 38 Amino Acid Domain in the N-terminal Thrombin Fragment of Human Osteopontin

Previous work from our laboratory demonstrates that the alpha(4)beta(1) integrin is an adhesion receptor for OPN and that alpha(4)beta(1) binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165-1174). The work presented here identifies two alpha(4)beta(1) binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial experiments, using wild-type OPN containing an RGD sequence or an OPN-RGE mutant, showed identical alpha(4)beta(1)-dependent cell adhesive activity. A strategy to localize alpha(4)beta(1) binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all binding activity was present within that portion of the molecule. Combined mutation and deletion analyses of this region revealed the involvement of dual alpha(4)beta(1) binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block alpha(4)beta(1)-dependent adhesion. The first peptide when coupled to Sepharose bound the alpha(4)beta(1) integrin directly whereas a mutated ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind. These data collectively demonstrate that dual alpha(4)beta(1) integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor.

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.