Recombinant Osteopontin Research Articles

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

Effect of ligand-receptor interaction of osteopontin-CD44 on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro.

To investigate the effect of ligand-receptor interaction of osteopontin (OPN)-CD44 on the expression of hyaluronic acid (HA) in the cultured human knee osteoarthritis (OA) chondrocytes via interfering the reaction between OPN and CD44 ligand-receptor. The OA chondrocytes and normal chondrocytes were obtained from knee joint cartilage tissues in the patients with knee OA and malignant tumor respectively. The normal chondrocytes and OA chondrocytes were detected and analyzed, and then the intervention analysis of OA chondrocytes was carried out. The OA chondrocytes were divided into 4 groups: a negative control group, which was cultured with complete medium without any molecular intervention reagent; an OPN intervention group, which was cultured with recombinant human OPN (rhOPN) for 24 hours; a CD44 blocking group, which were pretreated with CD44 receptor specific antagonist for 1 hour to block the binding of OPN-CD44, and then treated with rhOPN for 23 hours; a CD44 homotype group, which was pretreated with CD44 for 1 hour and then treated with rhOPN for 23 hours. In addition, the study for OPN-CD44 axis was also divided into 4 groups: an OA-negative control group (OA-NC group), a si-OPN intervention group, a rhOPN intervention group, and a rhOPN + CD44 antibody (Ab) group. Western blotting, real-time PCR, and enzyme linked immunosorbent assay (ELISA) were used to detect the protein and mRNA expression levels of OPN, CD44, hyaluronate synthase (HAS), and HA, respectively. The protein expression levels of OPN, CD44, and HAS1 and the secretion levels of HA in the OA chondrocytes were higher than those in the normal chondrocytes. Compared with the OPN intervention group, the expression levels of HAS1, HAS2, HAS3 and HA in the CD44 blocking group were lower than those in OPN intervention group (all P<0.05); but there was no significant difference in the expression levels of HAS1, HAS2, HAS3 and HA between the CD44 homotype group and the OPN intervention group (all P>0.05). The results of OPN-CD44 axis study showed that: compared with the OA-NC group, the expression of CD44 in the rhOPN intervention group was slightly lower, but the protein and mRNA levels of HAS1 were significantly increased (all P<0.05); compared with the OA-NC group, the expression of CD44 was up-regulated, but the protein and mRNA level of HAS1 were significantly inhibited in the si-OPN intervention group (all P<0.05); compared with the OA-NC group, the protein and mRNA levels of HAS1 in the rhOPN+CD44 Ab group were also significantly inhibited (all P<0.05). The OPN in OA chondrocytes can promote the expression of HAS1, and the OPN can stimulate the secretion of HAS and induce HA expression by reacting with CD44 ligand receptor. They constitute the axis of OPN/CD44/HAS1, which plays an important role in regulating the expression of HA in chondrocytes.

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.

Bacterial cellulose membrane conjugated with plant-derived osteopontin: Preparation and its potential for bone tissue regeneration

Bacterial cellulose membrane (BCM) has been recently recognized as a new generation of carbohydrate-based nanomaterial that possesses a great potential in tissue engineering applications. This research aims to develop an active non-resorbable guided tissue regeneration (GTR) membrane from BCM by conjugating with plant-derived recombinant human osteopontin (p-rhOPN), an economically produced and RGD-containing biomolecule. The BCM was initially grafted with poly(acrylic acid) (PAA) brushes to form poly(acrylic acid)-grafted BCM. Multiple carboxyl groups introduced to the BCM by PAA can serve as active anchoring points for p-rhOPN conjugation and yielded p-rhOPN-BCM. All chemically modified BCMs were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while their surface morphology was evaluated by field emission-scanning electron microscopy and atomic force microscopy analyses. The amount of p-rhOPN adhered on the membrane was quantified by enzyme-linked immunosorbent assay. The immunocytochemistry, two-stage quantitative real-time reverse transcriptase polymerase chain reaction and in vitro mineralization analyses strongly suggested that p-rhOPN-BCM could elicit biological functions leading to the enhancement of osteogenic differentiation of human periodontal ligament stem cells as effective as BCM conjugated with commercially available rhOPN from mammalian cells (rhOPN-BCM), suggesting its potential to be used as GTR membrane to promote bone tissue regeneration.

Fam20C-mediated phosphorylation of osteopontin is critical for its secretion but dispensable for its action as a cytokine in the activation of hepatic stellate cells in liver fibrogenesis.

Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.

Osteopontin-Enhanced Autophagy Attenuates Early Brain Injury via FAK-ERK Pathway and Improves Long-Term Outcome after Subarachnoid Hemorrhage in Rats.

Osteopontin (OPN) enhances autophagy, reduces apoptosis, and attenuates early brain injury (EBI) after a subarachnoid hemorrhage (SAH). A total of 87 Sprague–Dawley rats were subjected to sham or SAH operations to further investigate the signaling pathway involved in osteopontin-enhanced autophagy during EBI, and the potential effect of recombinant OPN (rOPN) administration to improve long-term outcomes after SAH. Rats were randomly divided into five groups: Sham, SAH + Vehicle (PBS, phosphate-buffered saline), SAH + rOPN (5 μg/rat recombinant OPN), SAH + rOPN + Fib-14 (30 mg/kg of focal adhesion kinase (FAK) inhibitor-14), and SAH + rOPN + DMSO (dimethyl sulfoxide). Short-term and long-term neurobehavior tests were performed, followed by a collection of brain samples for assessment of autophagy markers in neurons, pathway proteins expression, and delayed hippocampal injury. Western blot, double immunofluorescence staining, Nissl staining, and Fluoro-Jade C staining assay were used. Results showed that rOPN administration increased autophagy in neurons and improved neurobehavior in a rat model of SAH. With the administration of FAK inhibitor-14 (Fib-14), neurobehavioral improvement and autophagy enhancement induced by rOPN were abolished, and there were consistent changes in the phosphorylation level of ERK1/2. In addition, early administration of rOPN in rat SAH models improved long-term neurobehavior results, possibly by alleviating hippocampal injury. These results suggest that FAK–ERK signaling may be involved in OPN-enhanced autophagy in the EBI phase after SAH. Early administration of rOPN may be a preventive and therapeutic strategy against delayed brain injury after SAH.

Osteopontin attenuates early brain injury through regulating autophagy-apoptosis interaction after subarachnoid hemorrhage in rats.

AimTo determine the effect of osteopontin (OPN) on autophagy and autophagy‐apoptosis interactions after SAH.MethodsThe endovascular perforation model of SAH or sham surgery was performed in a total of 86 Sprague‐Dawley male rats. The temporal expressions of endogenous OPN and autophagy‐related proteins (Beclin 1, ATG5, LC3 II to I ratio) were measured in sham and SAH rats at different time points (3, 6, 12, 24, and 72 hours). Rats were randomly divided into three groups: Sham, SAH + Vehicle (PBS, phosphate‐buffered saline), and SAH + rOPN (5 μg/rat recombinant OPN). Neurobehavioral tests were performed 24 hours after SAH, followed by the collection of brain samples for assessment of autophagy and apoptosis proteins. These tests assessed whether an autophagy‐apoptosis relationship existed on the histological level in the brain.ResultsEndogenous OPN and autophagy‐related proteins all increased after SAH. rOPN administration improved neurological dysfunction, increased the expression of autophagy‐related proteins (Beclin 1, ATG5, LC3 II to I ratio) and antiapoptotic protein Bcl‐2, while decreasing the expression of proapoptotic proteins (cleaved Caspase‐3 and Bax). rOPN also regulated autophagy‐apoptosis interactions 24 hours after SAH.ConclusionrOPN attenuates early brain injury and inhibits neuronal apoptosis by activating autophagy and regulating autophagy‐apoptosis interactions.

Osteopontin promotes survival of intestinal intraepithelial lymphocytes and protects against colitis

Abstract Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells that reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes ranging from pathogen control to tissue stability. However, maintenance of IEL homeostasis is incompletely understood. In this report, we present evidence that osteopontin, a glycophosphoprotein with diverse roles in biomineralization, cell-mediated immunity, and inflammation, is important for maintaining normal levels of IEL. Mice in which the osteopontin gene (Spp-1) is disrupted present decreased levels of IEL subtypes, such as TCRαβ and TCRγδ IEL in the intestine, an effect not observed for lymphocytes in other immune compartments such as spleen or lamina propria, indicating an epithelium-specific effect. In vitro experiments show that mouse and human IEL survival is improved by culture with recombinant osteopontin. In vivo and in vitro IEL survival studies show that CD44, a ligand for osteopontin, is critical for the capacity of osteopontin to promote IEL survival. Adoptive transfer of total T cells (including Tregs) from wild type mice into Spp-1−/−Rag-2−/− mice results in the development of exacerbated intestinal inflammation, as compared with Rag-2−/− recipient mice. Further analyses show that osteopontin-deficiency results in decreased survival of Foxp3-expressing T cells. These findings support osteopontin’s critical role in IEL homeostasis and make it a potential target for curbing intestinal inflammation.

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics.

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood-brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.

An antibody-free sample pretreatment method for osteopontin combined with MALDI-TOF MS/MS analysis.

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 μg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

Plant-produced recombinant Osteopontin-Fc fusion protein enhanced osteogenesis

Osteopontin (OPN) plays an important role in the bone regeneration process. Previous investigation showed that recombinant human OPN was able to express in Nicotiana benthamiana leaves and induced the osteogenic related genes. Nevertheless, the purification of OPN from plant proteins with Ni affinity chromatography was still not effective enough. To improve the quality of protein expression and purification in plants, we constructed an Fc-based form of OPN. The complete OPN protein was fused to the human IgG1 Fc domain. Here, we showed that the plant-produced OPN-Fc increases the protein expression level and facilitates the purification of the recombinant protein. Our result showed that the plant-produced OPN-Fc can stimulate the expression of osteogenic related genes such as DMP1, OSX, and Wnt3a and also the calcium deposition in hPDL cells. These findings suggest that the plant-produced OPN-Fc has potential application in tissue engineering in the future.

Osteopontin potentiates PM-induced IL-1α and IL-1β production via the ERK/JNK signaling pathway

Ambient particulate matter (PM) poses a great threat to global health and contributes to pulmonary inflammation. However, the potential mechanism of PM-induced inflammation of the lung remains unclear. Osteopontin (OPN) is a multifunctional protein that reportedly regulates inflammatory responses in different diseases. Here, we explored the expression of OPN with PM exposure in vivo and in vitro and attempted to elucidate the regulatory role of OPN in PM-induced airway inflammation. Our results showed that PM exposure increased the expression of OPN in the bronchial epithelium, serum, and bronchoalveolar lavage fluid (BALF) of mice. Moreover, PM induced OPN expression in human bronchial epithelial cells (HBECs) in a dose and time-dependent manner. In vitro, inflammatory cytokines such as IL-1α and IL-1β were increased in HBECs with PM exposure via the ERK and JNK signaling pathways. Recombinant human OPN could potentiate PM-induced expression of IL-1α and IL-1β, while OPN siRNA could alleviate PM-induced inflammatory responses in HBECs. Furthermore, we showed that OPN regulated PM-induced inflammatory cytokines via the ERK and JNK pathways in HBECs. This study shows for the first time the positive effect of OPN on PM-induced airway inflammation and contributes to a better understanding of its potential mechanism of action.

LL-37 and its analog FF/CAP18 attenuate neutrophil migration in sepsis-induced acute lung injury.

Sepsis can result in acute lung injury. LL-37 is a small cationic host defense peptide involved in anti-inflammatory. In the current study, it was hypothesized that antimicrobial peptide LL-37 could play a protective role in attenuating the progression of sepsis-induced acute lung injury. Forty male C57BL/6 mice were induced into sepsis using cecal ligation and puncture, and subsequently administered with recombinant mouse osteopontin. Peptides LL-37, the LL-37 analog (FF/CAP18, called sLL-37), or normal saline was intravenously administered into septic mice for 20 hours. Then, proinflammatory cytokines (IL-6 and IL-1β), acute lung injury markers (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]), the neutrophil infiltration marker (myeloperoxidase [MPO]), and neutrophil infiltration were detected. Furthermore, the neutrophil migration and expression of migration-related factors (focal adhesion kinase [FAK], ERK, and P38) in differentiated HL-60 cells were detected. Septic mice had upregulated IL-6, IL-1β, ALT, AST, LDH, MPO, p-FAK, p-ERK, and p-P38, infiltrated neutrophils, and migrated neutrophil-like HL-60 cells. In contrast, the administration of peptide LL-37 and sLL-37 inhibited all these changes. Compared with septic mice, it was found that proinflammatory cytokines, lung injury markers, MPO, and infiltrated neutrophils decreased in mice treated with LL-37 and sLL-37. In addition, the migrated neutrophil-like HL-60 cells and activated p-FAK, p-ERK, and p-P38 proteins were suppressed by LL-37 and sLL-37 treatments. Peptide LL-37 and its analog sLL-37 attenuated the progression of sepsis-induced acute lung injury by inhibiting neutrophil infiltration and migration through the FAK, ERK, and P38 pathways.

OPN Maintains Leukemia Stem Cells Survival By Suppressing PUMA

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. In the era of chemotherapy, the cure rate of AML is 35 to 40% of adult patients who are 60 years of age or younger while it is only 5 to 15% of patients who are older than 60 years of age could achieve relative long term survival. Leukemia stem cells (LSCs) population is a small proportion in leukemia cells but plays an important role in initiation of AML and is the main cause of the recurrence after chemotherapy due to the ability of maintaining quiescence and other mechanisms to sustain survival. How to eradicate LSCs was investigated intensively during past years, but how LSCs maintain survival is still elusive. Here we show that osteopontin(OPN), a member of extracellular matrix protein, is essential for AML stem cells survival and could be a novel and potent therapeutic target for eradicating LSCs.We used RNA interference system to knock down OPN in a mouse model of MLL-AF9 induced AML. We examined the survival of AML mice and found that OPN knockdown (KD) group showed a significant prolongation of survival. Colony forming assay showed that OPN KD AML cells formed colonies in a lower frequency than that of control AML cells. Flow cytometry showed the frequency of LSC in KD AML cells was lower than that in control AML cells(0.04±0.02% vs 3.61±0.32%) and the median fluorescence density of c-Kit is lower in KD AML cells than of control AML cells(759.2±80.8 vs 1353.5±27.5). Brdu assay indicated an increased quiescent fraction in G0/G1 phases (64.9±8.9% vs 53.6±3.4%) of KD AML cells. The apoptosis of KD AML cells is increased than that in the control AML cells(14.82±3.4% vs 4.9±1.2%). Most interestingly, the change of phenotype is much more prominent in the LSCs population, ( comparing KD group and control group, frequency of G0/G1 in LSCs: 50.2±7.5% vs 26.4±3.7%; frequency of apoptotic cells in LSCs: 58.42±5.38% vs 7.45±1.57%). Limiting dilution assay confirmed that lower frequency of LSCs in the KD AML cells than in control(1/1204 vs 1/214). Thus we concluded that OPN plays a very important role in maintain survival of LSCs.To determine whether OPN knockdown could induce the same phenotype in human AML cells. We constructed OPN knockdown cell-line model by RNA interference system. Human AML cell-lines (SKM-1, ML2, OCI-AML3, SKM-1 FL3-ITD knock in) presented the same phenotype such as the increase of apoptosis and the decrease of the fraction of cells entering cell cycle. Then we performed Realtime PCR and western blot in mouse model to reveal the underlying mechanisms. Realtime PCR showed a significantly up-regulation of P16, P18, P21, which is related to the cell cycle arrest; western blot showed a significantly increase of PUMA and cleaved-caspase3, which indicated that knockdown OPN could promote apoptosis through repress the function of BCL2 by up-regulate PUMA in protein level. Interestingly, incubation of recombinant OPN protein with OPN KD cells did not rescue the phenotype suggesting that intracellular function of OPN in LSCs is important for its survival. The detailed mechanism of how OPN regulate PUMA is still under investigation.In our current study, it showed that OPN knock down can significantly prolong the survival of leukemic mice and decrease the leukemia burden by decreasing the stemness of LSC and promoting LSC apoptosis by inducing PUMA protein function. Thus it could be a good therapeutic target for eradicating LSCs in AML. DisclosuresNo relevant conflicts of interest to declare.

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates.

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019.

Surface-immobilized plant-derived osteopontin as an effective platform to promote osteoblast adhesion and differentiation.

In this report, recombinant human osteopontin synthesized in tobacco plants (p-rhOPN) is introduced as a potential bioactive molecule that can promote osteoblast adhesion and differentiation. A glass substrate (SiO2/Si-OH) grafted with poly(acrylic acid) (SiO2/Si-PAA) was prepared by surface-initiated reversible addition-fragmentation chain transfer polymerization and used as a carboxyl-rich platform for the chemical conjugation of p-rhOPN. The PAA grafting and subsequent p-rhOPN immobilization were confirmed by water contact angle, Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy analyses. Indirect ELISA quantification revealed that the p-rhOPN immobilization efficiency was above 95% and the surface coverage was a function of the p-rhOPN concentration. MC-3T3-E1 cells cultured on the SiO2/Si-PAA substrate immobilized with various concentrations (0.6-30 ng/mL) of p-rhOPN (SiO2/Si-p-rhOPN) exhibited superior cell spreading compared to those cultured on SiO2/Si-OH or gelatin-modified glass substrate (SiO2/Si-gelatin). Polymerase chain reaction analysis indicated that the SiO2/Si-p-rhOPN substrates with high level of immobilized p-rhOPN promoted MC-3T3-E1 cell differentiation, as demonstrated by the higher transcript expression levels of the osteogenic differentiation regulatory gene, Runt-related transcription factor 2, compared to cells cultured on SiO2/Si-OH or SiO2/Si-gelatin. Given that p-rhOPN can be more economically produced than the commercially available OPN derived from human or mammalian sources, then, together with its well-preserved biological function in spite of being chemically conjugated to the substrates, it is likely that p-rhOPN could be more broadly applied for the development of materials for bone tissue engineering with a promising medical and commercial value.

Anti‑osteopontin autoantibodies in various types of cancer.

The aberrant processing of biomolecules by cancer cells may give rise to autoimmune phenomena. The metastasis gene osteopontin is alternatively spliced only in transformed cells, and the variants promote tumor progression. They may also serve as tumor‑associated antigens, and their neutralization by autoantibodies could favorably affect prognosis. A competitive solid‑phase ELISA format was applied to determine reactivity toward recombinant osteopontin in human serum or plasma samples. Approximately 35% of thyroid cancer patients and 15% of other cancer patients were found to harbor autoantibodies directed to osteopontin variants, averaging 21% of patients across all cancers studied. The reactivity of the autoantibodies was consistent with the differential appearance of splice variants in individual malignancies. In thyroid cancer, autoantibodies were found to be more frequently associated with a milder form of the disease. The junctions of osteopontin splice variants produced by cancers represent tumor‑associated neo‑epitopes. Whereas the uniqueness of their sequences renders the acquisition of tolerance during immune maturation improbable, their immunogenicity is predicted to be low. The rare occurrence of antibodies to either osteopontin‑b or osteopontin‑c suggests that the breaking of tolerance to full‑length osteopontin is more prevalent in tumor autoimmunity than a reaction to a poorly immunogenic neo‑epitope.

HE4 suppresses the expression of osteopontin in mononuclear cells and compromises their cytotoxicity against ovarian cancer cells.

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4-mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual-specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up-regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN-inducible cytokines [interleukin (IL)-12 and interferon (IFN)-Ɣ]. Additionally, upon co-culture with PBMCs, HE4-silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN+ T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN-mediated T cell activation.

Osteopontin attenuates inflammation via JAK2/STAT1 pathway in hyperglycemic rats after intracerebral hemorrhage

Acute intracerebral hemorrhage (ICH) complicated by hyperglycemia is associated with aggravation of post-stroke inflammation, leading to exacerbation of brain edema and predicting poor neurological outcomes and higher mortality of patients. Osteopontin (OPN) is a neuroprotective glycoprotein, which is able to attenuate brain injury induced by hemorrhagic stroke. In the current study we investigated whether OPN will decrease the inflammatory post-ICH response as well as attenuate brain edema and neurological deficits in hyperglycemic rats. We employed a collagenase model of ICH on male Sprague-Dawley rats (n = 148) rats and 50% of Dextrose was injected intraperitoneally (i.p) 3 h after ICH (ICH + HG). Intranasal administration of recombinant OPN (rOPN) was performed 1 h after ICH. The development of brain injury was evaluated by brain water content (BWC) and neurological deficits, western blot and immunohistochemistry study. Small interfering ribonucleic acid (siRNA) for integrin-β1 receptor and a JAK2 agonist, Coumermycin A1 (C-A1), were used for detailed investigation of the molecular pathway. The administration of OPN (3 μg) significantly improved neurobehavior and increased expression of OPN and integrin-β1 receptor in the brain followed with decrease of neutrophil infiltration, JAK2, STAT1, TNF-a, IL-1b, MMP-9 and brain edema in the ICH + HG + OPN rats compared with ICH + HG rats. The effects of OPN were reversed by the intervention of intergrin-β1 siRNA and C-A1. In conclusion, rOPN attenuated ICH-induced brain inflammation in hyperglycemic rats, leading to attenuation of brain edema and improving neurological functions. Effects of rOPN were mediated at least partly by integrin-β1 induced inhibition of JAK2/STAT1 pathway.

Recombinant Osteopontin Improves Neurological Functional Recovery and Protects Against Apoptosis via PI3K/Akt/GSK-3β Pathway Following Intracerebral Hemorrhage

BackgroundThis study aimed to investigate the potential neuroprotective effect of recombinant osteopontin (r-OPN) on apoptotic changes via modulating phosphoinositide-3-kinase/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK-3β) signaling in a rat model of intracerebral hemorrhage (ICH).Material/MethodsWe subjected 10–12-week-old Sprague-Dawley male rats (n=120) to injection of autologous blood into the right basal ganglia to induce ICH or sham surgery. ICH animals received vehicle administration, r-OPN (4 μL/pup), or r-OPN combined with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (86 ng/pup) at 30 min after injury. Neurological scores and rotarod latencies were evaluated on days 1–5 post-ICH. Brain water content was evaluated on days 1–3 post-ICH. The number of apoptotic cells changes were evaluated by terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate-biotin nick-end labeling (TUNEL) and hematoxylin staining. Apoptosis-related proteins Bcl-2, Bax, and cleaved caspase-3 (CC3), and the phosphorylation levels of Akt and GSK-3β were assayed by Western blot.ResultsNeurological deficits, rotarod latencies, and brain water content following ICH were reduced in the r-OPN group compared to the vehicle group. r-OPN also attenuated cell death in ICH. Furthermore, treatment with r-OPN significantly increased p-Akt expression and decreased p-GSK-3β. These effects were associated with a decrease in the Bax/Bcl-2 ratio and the suppression of CC3 at 24 h after ICH. Importantly, all the beneficial effects of r-OPN in ICH were abrogated by the PI3K inhibitor wortmannin.Conclusionsr-OPN may provide a wide range of neuroprotection by suppressing apoptosis through the PI3K/Akt/GSK-3β signaling pathway after ICH.