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Research Thesis

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Research Thesis

  1. 1. 1 Examination of the Interaction of ADAM Proteins with Oxidized Phospholipids and their Role in Endothelial Inflammation Tridu Huynh, 1, 3 James R. Springstead,1 Sangderk Lee,2 Judith A. Berliner1,2,4 1 Department of Medicine, Division of Cardiology 2 Department of Pathology University of California – Los Angeles, Los Angeles, CA 90095, USA 3 Correspondence: tridu.huynh@gmail.com 4 Correspondence: jberliner@mednet.ucla.edu UID: 703-773-534
  2. 2. 2 SUMMARY Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and subsequent inflammation of the artery walls that can result in heart attacks and strokes. PAPC is one of the major phospholipids in low-density lipoprotein (LDL), and products of its oxidation (Ox-PAPC) interact and activate endothelial cells, which leads to the induction of chemokines, such as IL-8. IL-8 results in the migration and retention of monocytes into the subendothelial space, an initial step in atherogenesis. IL-8 induction is regulated by several pathways, one of which is the ADAM-mediated HBEGF-EGFR pathway. It has previously been shown that Ox- PAPC binds to several endothelial cell proteins, among which are some ADAMTS proteins. In this study, using Ox-PAPE-N-biotin, a biotinylated analog of Ox-PAPC, we present evidence that Ox-PAPC activates ADAM proteins, specifically ADAMTS1 and ADAMTS4, both of which have been implicated in IL-8 regulation, by covalently binding to them.
  3. 3. 3 INTRODUCTION Atherosclerosis, a chronic inflammatory disease of the artery wall, is the major cause of heart attacks and strokes, which are the leading causes of death in the United States (Heron, 2007). Atherosclerosis is characterized by the accumulation of lipids and fibrous debris in the subendothelial space of artery walls. Early atherosclerotic lesions consist of the formation of fatty streaks in arteries, which results from the accumulation of lipid-engorged macrophages, or foam cells, in the subendothelial space. Although fatty streaks are not clinically significant, they are the precursor to fibrous plaques, which arise from the migration of smooth muscle cells and the accumulation of lipid-rich necrotic debris in these now more advanced lesions. The final clinical complication of atherosclerosis is thrombosis, the formation of a blood clot inside a blood vessel as a result of the rupture of the unstable atherosclerotic lesion. Such blood clot can obstruct the blood flow through the circulatory system, resulting in a myocardial infarction or stroke (Lusis, 2000). Results from many clinical studies and animal models have shown that high levels of low-density lipoprotein (LDL), a fat carrier in the bloodstream, are strongly correlated with atherosclerotic development (Schwenke et al., 1989; Goldstein et al., 1977). More specifically, it was noticed that LDL lipids were oxidized in the subendothelial space of arteries after retention. These now called minimally modified LDL (MM-LDL) were seen to predict and accumulate in atherosclerotic lesions (Witztum et al., 1991). 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (PAPC) is one of the major phospholipids in LDL and cell membranes. It has previously been shown that products of oxidized PAPC (Ox-PAPC) are a major bioactive component of MM-LDL, and are present in atherosclerotic lesions (Leitinger et al., 1997; Watson et al., 1997). Ox-PAPC contributes to endothelial cell activation, a key initial event in atherogenesis, which enhances monocyte- endothelial interactions partly through the induction of chemokines, such as Interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) (Bobryshey et al., 2005). Previous research has
  4. 4. 4 shown that monocyte recruitment, retention and differentiation into the subendothelial space is an initial step in atherosclerotic plaque development. Upon entry, monocytes recruited at atherosclerotic lesions differentiate into macrophages that take up lipids until they eventually become lipid-laden foam cells that contribute to the formation of the fatty streak (Insull et al., 2009). IL-8 is regulated by many pathways, one of which is the heparin-binding epidermal growth factor and epidermal growth factor receptor (HBEGF-EGFR) pathway. We have shown in previous studies that Ox-PAPC activates certain ADAM proteins (a disintegrin and metalloproteinase), and that such activated ADAMs process HBEGF on the cell surface. The soluble HBEGF ligand then binds to the EGFR, leading to IL-8 induction in the cell (Lee et al., 2012; Figure 1). Using Ox-PAPE-N-biotin (Ox-PNB), a biotinylated analog of Ox-PAPC with identical biological properties, it has previously been demonstrated that Ox-PAPC binds to several endothelial cell proteins (Gugiu et al., 2008).Furthermore, we previously showed that Ox-PAPC covalently binds to cysteine residues on specific ADAMs in endothelial cells (Lee et al., 2012). We hypothesize that binding of Ox-PAPC activates the ADAMs, which would then result in an induction of IL-8 in endothelial cells through the aforementioned ADAM-mediated HBEGF- EGFR pathway. This study focuses on the interaction between the metalloproteinases (MPs) ADAMTS1 and ADAMTS4 (a disintegrin and metalloproteinase with thrombodspondin motifs) and Ox- PAPC. ADAMTS4 is the subject of current study because it was previously shown to be involved in IL-8 regulation by Ox-PAPC in past silencing studies (Lee et al., 2012). ADAMTS1 is the subject of current study because it is known to cleave VEGFR2, which plays a role similar to EGFR in IL-8 regulation. The ADAMTS are a group of proteases that are found both in mammals and invertebrates. They are extracellular, multi-domain enzymes that have several known functions, one of which is the cleavage of matrix proteoglycans aggrecan and versican
  5. 5. 5 (Porter et al., 2005). Aggrecan is an extracellular matrix proteoglycan that was used in past and present studies to assay the activity of certain ADAMTS proteins. In this study, using Ox-PNB, we present evidence that Ox-PAPC activates ADAMTS1 and ADAMTS4’s enzymatic activity and that it covalently binds to them. RESULTS Ox-PAPC Activates ADAM Proteins To determine whether Ox-PAPC activates ADAM proteins’ enzymatic activities, we measured the processing of fluorogenic ADAM substrate. Human Aortic Endothelial Cells (HAECs) were treated with either no Ox-PAPC or 50ug/mL of Ox-PAPC and substrate cleavage was assayed at various time points (Figure 2A). Ox-PAPC is seen to clearly increase the activity of ADAM proteins over time. To further prove the point, HAECs were treated with varying concentrations of GM6001 or Batimastat (matrix metalloproteinase inhibitors) for 4 hours, and the amount of ADAM cleavage was assayed through fluorescence quantification, again (Figure 2B). Increasing concentration of GM6001 or Batimastat were seen to inhibit Ox-PAPC’s activation of ADAM proteins. Ox-PAPC Activates Aggrecanases, ADAMTS1 and ADAMTS4 being two of them To hone in on a specific subset of ADAM proteins for further study, exogenous aggrecan was used as a substrate to determine whether or not aggrecanases are a subset of ADAM proteins that undergo activation in the presence of Ox-PAPC. Also, as previously mentionned, we previously showed that ADAMTS1 and ADAMTS4 are implicated in IL-8 regulation through Ox- PAPC, both of which are known aggrecanases (Boeuf et al., 2012). Western blot analysis of HAECs with aggrecan added in Ox-PAPC or control condition showed degradation of aggrecan as early as 1 hour (Figure 3A). This show that Ox-PAPC leads to activation of aggrecanase(s).
  6. 6. 6 HAECs with aggrecan added were then transfected with either ADAMTS4, ADAMTS1 or control and treated with either no Ox-PAPC or 50ug/mL of Ox-PAPC. In this experiment, the control condition consisted only of transfection reagent. Future experiments will consist of transfection with a plasmid lacking an ADAM protein as a better control. Ox-PAPC is seen to increase cleavage of aggrecan in both ADAMTS4 and ADAMTS1 transfected cells as well as untransfected cells (Figure 3B). This shows that Ox-PAPC activates enzymatic activities of ADAMTS4 and ADAMTS1 specifically. Ox-PAPC Demonstrates Specificity of Binding Given the chemically reactive nature of Ox-PAPC, it is plausible that it could have demonstrated opportunistic binding to a variety of molecules with no relevance to the model under study. To address this concern, HAECs were treated with different doses of Ox-PNB (10, 7, 4, 1, and 0 ug/mL) for four hours. Western blot analysis was then performed to visualize Ox-PNB bound proteins using streptavidin-HRP. The untreated condition revealed a couple of non-specific bands that represent endogenous proteins with avidin-binding properties (Cauli et al., 1994). A noticeable band was detected around 90kDa, with binding seen at concentrations as low as 1ug/mL (Figure 4). This suggests that Ox-PNB has a higher binding affinity for specific proteins. Ox-PAPC Binds to ADAMTS4 and ADAMTS1 To test the hypothesis that ADAMTS4 and ADAMTS1 are enzymatically activated through covalent binding to Ox-PAPC, human embryonic kidney 293 (HEK293) cells were transfected with either ADAMTS4-HA or ADAMTS1-HA, treated with 50ug/mL of either unoxidized PNB or Ox-PNB for 30 minutes, immunoprecipitated with streptavidin beads, and blotted with anti-HA- HRP. ADAMTS4 and ADAMTS1 have an expected molecular weight of 90 and 105kDa, respectively. There is a clear increase in band intensity in the according bands for both ADAMTS1 and ADAMTS4 going from treatment with PNB to treatment with Ox-PNB,
  7. 7. 7 suggesting that Ox-PNB, and therefore Ox-PAPC, binds to ADAMTS4 and ADAMTS1. However, the increase in band intensity is much greater for ADAMTS4 than ADAMTS1, suggesting that Ox-PAPC binds to ADAMTS4 more strongly (Figure 5). Ox-PAPC Promotes Cleavage of ADAMTS4 into Mature Form HEK293 cells were transfected with either ADAMTS1-HA or ADAMTS4-HA, treated with either phosphate buffered saline (PBS) or 50ug/mL of Ox-PAPC for an hour, immunoprecipitated with anti-HA beads, and blotted with anti-HA-HRP. There is a clear increase in band intensity for what is supposedly the cleaved, mature form of ADAMTS4 around 68kDa going from PBS to Ox-PAPC treatment. On the other hand, ADAMTS1’s cleaved, mature form around 85kDa does not seem to show such an increase (Figure 6). This suggests that Ox-PAPC promotes cleavage of ADAMTS4 into its active, mature form, but that ADAMTS1 does not undergo the same process. PCSK3 is Implicated in IL-8 Regulation by Ox-PAPC Given the results obtained in figure 5, we naturally became interested in the mechanism by which Ox-PAPC might lead to increased production of the mature form of ADAMTS4. Proprotein convertase subtilisin/kexin (PCSK) is a family of enzymes that perform cleavage and conversion of immature, target proteins into their biologically active forms (Turpeinen et al., 2011). PCSK3 (FURIN) is known to proteolytically process pro-ADAMTS4 into its mature form. We then first tested whether or not PCSKs had a role in IL-8 regulation by Ox-PAPC using silencing techniques. HAECs were transfected with either scrambled siRNA or one of two siRNAs against PCSK3. The second siRNA against PCSK3 resulted in a 40-50% knockdown of the protein with a corresponding ~30% knockdown of IL-8 induction in the cells (Figure 7). This is modest evidence that PCSKs might play a role in IL-8 regulation by Ox-PAPC.
  8. 8. 8 DISCUSSION This study provides evidence for the activation of ADAM proteins’ enzymatic activities through Ox-PAPC, specifically the aggrecanases ADAMTS4 and ADAMTS1, which we have previously shown to be implicated in IL-8 upregulation in endothelial cells by Ox-PAPC. Furthermore, using Ox-PNB, we show that Ox-PAPC clearly binds to ADAMTS4, with modest if not negligible binding to ADAMTS1 (Figure 5). Taken together, several hypotheses can be put forward as to how Ox-PAPC activates ADAMTS’s enzymatic activity. Other groups have shown that covalent interaction of metalloproteinases (MPs) with electrophiles caused enhancement of enzyme activity (Rajagopalan et al., 1996). A plausible mechanism of activation could be through Ox-PAPC displacing what is known as the “cysteine switch” from the zinc-containing catalytic domain of the MP. The cysteine switch is a cysteine-containing consensus sequence in the N-terminal pro-peptide domain that coordinates with the zinc ion in the catalytic site. The MP’s activity is suppressed as a result of zinc-cysteine coordination and pro-peptide domain occlusion of the active site (Rosenblum et al., 2007). This mechanism is of particular interest and the subject of further study to us because we previously showed that Ox-PAPC binds to cysteine residues in some ADAM and ADAMTS proteins, ADAMTS4 being one of them (Lee et al., 2012). Determination of the specific cysteines bound by Ox-PAPC is the subject of further study. Mutation of putative cysteine binding sites on ADAMTSs in an attempt to determine actual binding sites as well as to confirm whether binding is the actual mechanism of ADAMTS activation are the aims of future studies. The results of figure 6 were unexpected and hint at a possible involvement of PCSKs in IL-8 regulation by Ox-PAPC. There is a clear increase in what seems to be the cleaved, mature form of ADAMTS4 going from control to Ox-PAPC condition, showing that Ox- PAPC leads to activation of ADAMTS4’s processing. The same cannot be said of ADAMTS1, however, suggesting that ADAMTS1 does not undergo the same process. We hypothesized that Ox-PAPC binding to ADAMTS4 leads to a conformational change that would predispose it to
  9. 9. 9 processing by PCSK3 (FURIN), which is known to process pro-ADAMTS4 into its mature form (Wang et al., 2004). Silencing PCSK3 resulted in a modest (~30%) reduction in IL-8 levels in HAECs (Figure 7). The low impact of PCSK3’s silencing on IL-8 could be attributed to the fact that the silencing only reduced PCSK3’s level by 40-50%. It could also be due to the fact that IL- 8 is regulated by many pathways. Nevertheless, PCSK3 still shows some evidence of being involved in IL-8 upregulation by Ox-PAPC. Replication of PCSK3’s silencing is required to firmly determine that. We would also broaden our silencing study to other members of the PCSK family that might be involved in processing of ADAM proteins involved in IL-8 regulation by Ox- PAPC. EXPERIMENTAL PROCEDURES Preparation of Ox-PAPC PAPC (1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine) was purchased from Avanti Polar Lipids and was oxidized by exposure to air for 48 hrs. Oxidation was monitored by electrospray ionization-mass spectrometry (ESI-MS). PAPE-N-biotin synthesis A solution 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylethanolamine (PAPE) in dry dichloromethane was added drop-wise to a magnetically stirred solution of biotin, dicyclohexylcarbodiimide, and dimethylaminopyridine under argon at room temperature. The solution was mixed for 12 h at room temperature. The solvent was evaporated and the lipid was separated by reverse-phase high-performance liquid chromatography (HPLC) with ESI-MS detection in negative mode to produce 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphatidyl-(N- biotinylethanolamine) (PAPE-N-biotin). Cell Culture and Treatment
  10. 10. 10 Plates for Human Aortic Endothelial Cells (HAECs) or Human Embryonic Kidney 293 (HEK293) cells were coated with 0.1% gelatin-PBS. HAECs were cultured in MCDB-131 complete media (VEC technologies) alone or M199 medium supplemented with 20% FBS (Hyclone), 100U/mL penicillin, 100ug/mL streptomycin, 1mmol/L sodium pyruvate, 65ug/mL heparin (Sigma), and 50ug/mL endothelial cell growth supplement (ECGS) (BD Biosciences). HEK293 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 4.5 g/L glucose supplemented with 10% FBS (Hyclone), 100U/mL penicillin, 100ug/mL streptomycin, 1mmol/L sodium pyruvate. Ox-PAPC in chloroform (stock: 10mg/ml) was dried to a lipid residue and resuspended in M199 medium plus 1% FBS for cell treatment. Generally, cells were changed to M199 medium containing 1% serum for 30min before cell treatment. Cells were then incubated with or without Ox-PAPC in medium containing 1% serum. ADAM Substrate Cleavage Assay The activity of endogenous ADAMs in HAECs were determined using a fluorogenic ADAM substrate (Enzo BML-P235, Dabcyl-Leu–Ala-Gln–Ala-Homophe–Arg-Ser—Lys[5-FAM]-NH2). The product formation was determined by fluoroscence measurement using excitation at 485 nm and emission at 520 nm. Transfection of Plasmids or siRNAs 90% and above confluent cells were treated with plasmid or siRNA complexes with Lipofectamine 2000 (Invitrogen) for 4-6 hours in OPTIMEM media (Invitrogen) with fungizone at 37°C. The OPTIMEM media was then removed, washed with 1x PBS without calcium and magnesium, and replaced with 4.5g/L DMEM with 10% FBS media. Cells were used for experiments after 2 days of cell growth. The specific silencing of target genes was confirmed by qRT-PCR and Western blotting.
  11. 11. 11 Immunoprecipitation Anti-HA resins or Neutravidin beads (Roche) were used for immunoprecipitation. 1mL of lysate was mixed with 50uL of either beads in 1.5mL eppendorf tubes. The tubes were then sealed and incubated with gentle-end-over-end mixing in 4°C room overnight. Following the incubation, the lysate was centrifuged at 4,000g and the supernatant was removed. Resins were washed with 500uL of Tris-buffered saline containing 0.1% Tween 20 (TBST) three times. 45uL of sample buffer consisting of 2x SDS sample buffer with β-mercaptoethanol in a 19:1 ratio was added to the tubes and then boiled for 5 minutes. The tubes were then centrifuged for 2 minutes at 4000rpm and the eluent was collected and ready to be loaded on a gel. Western Blotting Laemmeli buffer (2x, Bio-rad) containing both protease and phosphatase inhibitors and PMSF (1mM) was used for protein-samples preparation for SDS-PAGE. The samples were loaded onto wells of a 4-20% Tris-glycine SDS gel (NuGel). Blots were transferred overnight. The blots were incubated with primary and secondary antibodies in 5% milk or 1% BSA in TBST. They were then developed and analyzed using enhanced chemiluminescence (ECL) prime kit (Amersham). VersaDoc Imaging System (BioRad) and Quantity One® program were used for image acquirement and band density analysis. Quantitative Real-Time PCR (qRT-PCR) Total RNAs and cDNAs were isolated and prepared using RNA extraction and cDNA synthesis kits from Bio-Rad. SYBR® green master mixture and PCR amplification system from Roche Diagnostics were used for PCR amplification and quantification procedure. The transcriptional level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined for each cDNA normalization.
  12. 12. 12 REFERENCES Bobryshev, Y.V. (2005). Monocyte recruitment and foam cell formation in atherosclerosis. Micron. 37, 208-222. Boeuf, S., Graf, F., Fischer, J., Moradi, B., Little, C.B., Richter, W. (2012). Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells. Eur. Cell. Mater. 4, 320-32. Cauli, A., Yanni, G., Panavi, G.S. (1994). Endogenous avidin-binding activity in epithelial cells of the ducts of the human salivary glands. Clin. Exp. Rheumatol. 12, 45-7. Goldstein, L.J., Brown S.M. (1977). The low-density lipoprotein pathway and its relation to atherosclerosis. Annu. Rev. Biochem. 46, 897-930. Gugiu, G.B., Mouillesseaux, K., Duong, V., Herzog, T., Hekimian, A., Koroniak, L., Vondriska, T.M., Watson, A.D. (2008). Protein targets of oxidized phospholipds in endothelial cells. J. Lipid Res. 49, 510-520. Heron, M. (2011). Deaths: leading causes for 2007. Natl. Vital Stat. Rep. 59, 1-95. Insull, W. (2009). The Pathology of Atherosclerosis: Plaque Development and Plaque Responses to Medical Treatment. Am. J. Med. 122, S3-S14. Lee, S., Springstead, J.R., Parks, B.W., Romanoski, C.E., Palvolgyi, R., Ho, T., Nguyen, P., Lusis, A.J., Berliner, J.A. (2012). Metalloproteinase processing of HBEGF is a Proximal event in the Response of human aortic endothelial cells to oxidized phospholipids. Arterioscler. Thromb. Vasc. Biol. 32, 1246-1254. Leitinger, N., Watson A.D., Faull K.F., Fogelman A.M., Berliner J.A. (1997). Monocyte binding to endothelial cells induced by oxidized phospholipids present in minimally oxidized low density lipoprotein is inhibited by a platelet activating factor receptor antagonist. Adv. Exp. Med Biol. 433, 379-382. Lusis, A.J. (2000). Atherosclerosis. Nature 407, 233-41. Porter, S., Clark, I.M., Kevorkian, L., Edwards, D.R. (2005). The ADAMTS metalloproteinases. Biochem. J. 386, 15-27. Rajagopalan, S., Meng, X.P., Ramasamy, S., Harrison, D.G., Galis, Z.S. (1996). Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J. Clin. Invest. 98, 2572-9. Rosenblum, G., Meroueh, S., Toth, M., Fisher, J.F., Fridman, R., Mobashery, S., Sagi, I. (2007). Molecular structure and dynamics of the stepwise activation mechanism of a matrix metalloproteinase zymogen: challenging the cysteine switch dogma. J. Am. Chem. Soc. 129, 13566-74. Schwenke, D.C., Carew, T.E. (1989). Initiation of atherosclerotic lesions in cholesterol-fed rabbits. Focal increases in arterial LDL concentration precede development of fatty streak lesions. Arterioscler. Thromb. Vasc. Biol. 9, 895-907.
  13. 13. 13 Springstead, J.R., Gugiu, B.G., Lee, S., Cha, S., Watson, A.D., Berliner, J.A. (2012). Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function. J. Lipid Res. 53, 1304-15. Turpeinen, H., Raitoharju, E., Oksanen, A., Oksala, N., Levula, M., Lyytikäinen, L.P., Järvinen, O., Creemers, J.W., Kähönen, M., Laaksonen, R., et al. (2011). Proprotein convertases in human atherosclerotic plaques: the overexpression of FURIN and its substrate cytokines BAFF and APRIL. Atherosclerosis 29, 799-806. Wang, P., Tortorella, M., England, K., Malfait, A.M., Thomas, G., Arner, E.C., Pei, D. (2004). Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (aggrecanse-1) in the trans-golgi network. J. Biol. Chem. 279, 15434-40. Watson, A.D., Leitinger, N., Navab, M., Faull, K.F, Horkko, S., Witztum, J.L., Palinski, W., Schwenke, D., Salomon, R.G, Sha, W., et al. (1997). Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J. Biol. Chem. 272, 13597-13607. Witztum, J.L., Steinberg, D. (1991). Role of oxidized low density lipoprotein in atherogenesis. J. Clin. Invest. 88, 1785–92. Yeh, M., Leitinger, N., De Martin, R., Onai, N., Matsushima, K., Vora, D.K., Berliner, J.A., Srinivasa, T.R. (2001). Increased Transcription of IL-8 in Endothelial Cells Is Differentially Regulated by TNF- α and Oxidized Phospholipids. Arterioscler. Thromb. Vasc. Biol. 21, 1585- 91. FIGURE LEGENDS Figure 1. Hypothesized induction of IL-8 expression through Ox-PAPC pathway. Figure 2. Ox-PAPC activates ADAM proteins. HAECs in 50mM Tris – pH 7.5 and 100mM NaCl buffer with 8uM of ADAM fluorogenic substrate (BML-P235 from Enzo). (A) Cells were treated with either no Ox-PAPC or 50ug/mL Ox-PAPC. Cleavage of the fluorogenic substrate was assayed at various time points as described in the Methods section (30, 60, 90, 120, 180, 240 mins).
  14. 14. 14 (B) Cells were treated with either no Ox-PAPC or 50ug/mL Ox-PAPC and varying concentrations of GM6001 or Batimastat (matrix metalloproteinase inhibitors) for 4 hours and the amount of ADAM cleavage was assayed through quantification of fluorescence. Figure 3. Ox-PAPC activates Aggrecanases. (A) HAECs in 50mM Tris – pH 7.5, 100 mM NaCl and 10mM calcium buffer with or without 50ug/mL Ox-PAPC. Exogenous aggrecan fragments were added to the cells due to the large size of endogenous aggrecan, and supernatant was collected at different time points (1, 2, 4 hours and overnight). Western blot analysis with anti-aggrecan shows that Ox-PAPC leads to degradation of aggrecan as early as 1 hour. (B) HAECs transfected with either vehicle or ADAMTS4 or ADAMTS1, treated with no Ox- PAPC or 50ug/mL of Ox-PAPC, and blotted with anti-aggrecan. Top band represents undigested aggrecan fragments, bottom band represents digested fragments. Figure 4. Ox-PNB demonstrates specificity of binding. HAECs treated with different doses of Ox-PNB for 4 hours. Western blot analysis with streptavidin-HRP was performed to visualize Ox-PNB bound proteins. A couple of non-specific avidin-binding proteins can be seen in the untreated lane. A band around 90kDa can be seen at concentrations as low as 1ug/mL. Figure 5. Ox-PAPC binds to ADAMTS4 and ADAMTS1. HEK293 cells transfected with ADAMTS4 or ADAMTS1, treated with 50ug/mL of PNB or Ox- PNB for 30 minutes, immunoprecipitated with streptavidin resins, and blotted with anti-HA-HRP. Arrows represent the size of each ADAMTS protein. Figure 6. Ox-PAPC promotes cleavage of ADAMTS4 into mature form.
  15. 15. 15 HEK293 cells transfected with ADAMTS4 or ADAMTS1, treated with PBS or Ox-PAPC for an hour, immunoprecipitated with anti-HA resins, and blotted with anti-HA-HRP. Arrows indicate the size of each ADAMTS’s mature size. Figure 7. PCSK3 is involved in IL-8 regulation by Ox-PAPC HEK293 cells transfected with PCSK3 siRNA or scrambled for 4 hours and grown in VEC media for 3.5 days before treatment with Ox-PAPC for 4 hours. Values normalized to GAPDH levels. siFURIN2 knocked down PCSK3 expression by 40-50% with a corresponding 30% knockdown of IL-8 induction. .
  16. 16. 16 FIGURE 1
  17. 17. 17 FIGURE 2 A 0 5000 10000 15000 20000 25000 30 60 90 120 180 240 Fluorescence(485/530nm) Incubation time (min) ADAM Activity in HAECs control Ox50 0 5000 10000 15000 20000 25000 30000 502512.56.253.1250 Fluorescence(485/530nm) GM6001 ADAM Activity with GM6001 Ox50 media 0 5000 10000 15000 20000 25000 30000 50025012562.531.250 Fluorescence(485/530nm) Batimastat ADAM Activity with Batimastat Ox50 media
  18. 18. 18 FIGURE 3 A B
  19. 19. 19 FIGURE 4
  20. 20. 20 FIGURE 5
  21. 21. 21 FIGURE 6
  22. 22. 22 FIGURE 7

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