Intracellular protein phosphorylation in adherent U937 monocytes mediated by various culture conditions and fibronectin-derived surface ligands

Chen, Xiuxu; Zuckerman, Sean T.; Kao, Weiyuan John

doi:10.1016/j.biomaterials.2004.04.002

Cited by 13 publications

(8 citation statements)

References 35 publications

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“…Although a number of polymers have been designed and investigated for biomedical and pharmaceutical applications1–4 poly (3‐hydroxybutyrate ‐co ‐3‐hydroxyvalerate) (PHBV) is still under extensive investigation because of its biologically favorable properties. PHBV is a natural biodegradable polymer that recently has received attention in the field of bone tissue engineering 5–9. The interest in using PHBV for bone tissue engineering applications is in part due to the reason that PHBV can be readily tuned to achieve desired mechanical properties and degradation rate 10–12.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the biological responses of osteoblast‐like UMR‐106 cells to the engineered porous PHBV matrix

Liu

Raghavan

Stubbs

2006

J Biomedical Materials Res

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Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been investigated for biomedical applications due to its many biologically favorable properties. However, to explore its application in bone tissue engineering, the poorly bioactive surface property of PHBV must be improved. To engineer PHBV to achieve a biologically active surface, in this study each porous PHBV matrix was prepared by solute leaching of salt/PHBV cast film and was treated with ozone followed by dip coating with type I collagen. The biological responses of osteoblast-like UMR-106 cells after being grown on the engineered PHBV matrix were evaluated. Confocal microscopy and the MTT assay were used to map and quantify the viable cell proliferation on the PHBV matrix, respectively. The cells were cultivated in osteogenic media containing beta-glycerophosphate and later stained with alizarin red to visualize mineralization of the matrix. RNA was extracted from the UMR-106 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to detect expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a house keeping gene) and bone sialoprotein (BSP) (marker of the osteoblastic phenotype). The results showed that the UMR-106 cells after cultivation on the engineered PHBV matrix retained the osteoblastic phenotype characteristics, indicating that the porous PHBV matrix after ozone treatment and collagen dip coatings are a promising scaffold for bone tissue engineering applications.

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Section: Introductionmentioning

confidence: 99%

Evaluation of the biological responses of osteoblast‐like UMR‐106 cells to the engineered porous PHBV matrix

Liu

Raghavan

Stubbs

2006

J Biomedical Materials Res

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“…Mass spectrometry coupled to HPLC was used to identify intracellular proteins expressed by adherent U937 macrophages in response to various culture conditions. These protein identities may provide insight into the previously observed densitometric trends shown in Table 1 [10].…”

Section: Comparison Of Nanospray Lc/ms Results Based On Previous Densmentioning

confidence: 71%

“…However, more work resolving the actual signaling cascades is needed to fully understand any signaling shifts and their implications regarding the host inflammatory response. Table 1 Differential expression of various intracellular proteins in adherent U937 on protein-modified TCPS treated with or without AG18 as assayed by previous study [10] Comparison of ~65/70 kDa proteins from adherent U937 on PBS treated TCPS with 0 or 40 μM AG18 Summary of conditions for elucidating the effect of varying AG18 concentrations Summary of conditions for analyzing the effect of surface-adsorbed ligands x-Samples obtained.…”

Section: Discussionmentioning

confidence: 99%

“…The IP beads and proteins were incubated at ~90°C for 10 min before SDS-PAGE on Tris-HCl gels (10% resolving, 4% stacking). The gels were run at 84 V for 30 min during the stacking gel and 129 V for 1 h 45 min through the resolving gel [10]. Under the present analysis scheme, one sample for protein analysis requires more than 25 million cells.…”

Section: Preparation Of Cell Lysates and Immunoprecipitationmentioning

confidence: 99%

“…Densitometry was performed on the gels after staining with Coomassie Brilliant Blue dye to identify bands showing up or down regulated protein expression. Various surface associated ligands and AG18 concentrations affected the expression levels of a set of proteins ranging from ~200 to ~23 kDa [10] (Table 1). Western blotting and mass spectrometry are two methods for protein identification in proteomics study [11,12].…”

Section: Introductionmentioning

confidence: 99%

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Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages

Zuckerman

Kao

2006

Biomaterials

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Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from approximately 200 to approximately 23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at approximately 52, approximately 42 and approximately 23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 microm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.

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LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface‐adsorbed fibronectin‐derived peptides

Zuckerman

Kao

2007

J Biomedical Materials Res

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The extent and duration of the host response determines device efficacy, yet the mechanism is poorly understood. U937 promonocytic cells were cultured on peptide-adsorbed tissue-culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine-phosphorylated proteins were chosen based on physiological significance and previous densitometry results, which identified a set of proteins ranging from approximately 200 to approximately 23 kDa showing altered phosphorylation levels in response to various surface-adsorbed ligands and phosphorylation inhibitor AG18. Although LC/MS has been used for nearly a decade, its application to the field of biomaterials is relatively novel. Twelve intracellular proteins identified by nanospray LC/MS are potentially related to the host response. Eight of the twelve proteins are related to the cytoskeleton including: moesin, heat shock protein 90beta, alpha-tubulin, elongation factor 1alpha, beta actin, vimentin, plasminogen activator inhibitor 2, and heterogeneous ribonuclear protein A2. The remaining four proteins: high mobility group box 1, caspase recruitment domain 5, glycoprotein 96, and heterogeneous nuclear ribonucleoprotein D0 modulate inflammation. The specific effect each peptide has upon modulating the phosphorylation state of these proteins cannot be determined from this work; however, 12 viable targets have been identified for further investigation into the role each plays in the surface-mediated monocyte response.

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Intracellular protein phosphorylation in adherent U937 monocytes mediated by various culture conditions and fibronectin-derived surface ligands

Cited by 13 publications

References 35 publications

Evaluation of the biological responses of osteoblast‐like UMR‐106 cells to the engineered porous PHBV matrix

Evaluation of the biological responses of osteoblast‐like UMR‐106 cells to the engineered porous PHBV matrix

Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages

LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface‐adsorbed fibronectin‐derived peptides

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