siRNA to ELK1 inhibits PRPRZ1 activation.

<p>(A) Immunoblotting analysis for ELK1, β-Actin, and GAPDH in HEK293T cells transfected with ELK1 scrambled siRNA (60 nM), β-Actin siRNA (60 nM), or ELK1 siRNA (60 nM). (B) ELK1 mRNA as determined by quantitative RT-PCR from HEK293T cells 48 hours after transfection with ELK1 scrambled siRNA, β-Actin siRNA, or ELK1 siRNA. (C) β-Actin mRNA under similar conditions. (D) PTPRZ1-250 promoter activity in HEK293T cells following transfection with HIF-2α plasmid alone or HIF-2α plasmid and either scrambled siRNA, or ELK1 siRNA. Data is presented as fold induction over vector control after normalization to β-gal. Bars represent mean and standard deviation of 3 determinations.</p

Comparison of the activation of truncated forms of PTPRZ1 luciferase reporter by HIF-1α and HIF-2α and their degradation-resistant forms.

<p>(A) Hep3B or (B) HEK293T cells were co-transfected with 300 ng of each PTPRZ1 promoter and 50 ng of an internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, drHIF-1α, HIF-2α, drHIF-2α, or pcDNA3.1control. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Figure 2</a>.</p

HIF-1 and HIF-2 bind to PTPRZ1 oligonucleotide probes containing HRE4.

<p>DIG-labeled synthetic oligonucleotide containing HRE4 was incubated with nuclear extracts (NE) from normoxic (N), 16 hour hypoxic (H) or HIF-transfected (HIF-1α or HIF-2α) HEK293T cells and analyzed on a non-denaturing polyacrylamide gel. Where indicated, unlabeled wild type (WT) or mutant (Mut) oligonucleotides (at 50x and 100x of the labeled probe) were added to the binding reaction. Protein-DNA complexes were separated, blotted to a nylon membrane, and probed with anti-digoxigenin antibody conjugated to alkaline phosphatase. Comp denotes unlabeled probe used for binding competition. The sequence of the probe and WT and Mut competing oligonucleotides used is shown at the bottom. WT HRE4 sequence is underlined, and nucleotide changes in the Mut sequence are shown in bold. The positions of the DIG-labeled HIF complexes and free probe are indicated with arrows.</p

Role of EBS4 in the activation of PTPRZ1 promoter by HIF-2α.

<p>(A) Effects of EBS4 or EBS5 deletion on the response of the PTPRZ1 promoter to HIF-2α. HEK293T cells were co-transfected with 300 ng of PTPRZ1-250WT, EBS4D, or EBS5D promoters and 50 ng of a internal β-gal control plasmid in the presence of 250 ng of an expression plasmid encoding HIF-1α, HIF-2α or pcDNA3.1 empty control vector. Results are expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009641#pone-0009641-g002" target="_blank">Fig 2</a>. (B). Binding of HIF-2α and HIF-1α to the PTPRZ1 promoter in the region near EBS4, EBS5, HRE4, and HRE5 <i>in vivo</i>. The chromatin immunoprecipitation assay was performed with HEK293T cells transfected with HIF-1α or HIF-2α respectively. Pre-cleared chromatin was immunoprecipitated with anti-HIF-2α or anti-HIF-1α antibody or normal rabbit IgG. After reversal of cross-linking, the DNA was analyzed by PCR. The primer set for PCR were designed to cover the EBS4, EBS5, HRE4, and HRE5 sites (C) Binding of ELK1 to the PTPRZ1 promoter. Experiment performed as in 7B except that the HEK293T cells were transfected with an Elk-1 expression vector and anti-ELK1 antibody or normal rabbit IgG was utilized.</p

PTPRZ1 luciferase promoter constructs showing the location of the potential hypoxia response elements (HRE) and Ets binding sequences (EBS).

<p>Each HRE is denoted as a square and each EBS as a plus sign. The TATA box and the ATG start site are indicated in the PTPRZ1-250 promoter. Each promoter construct extends 57 bp into the PTPRZ1 coding region prior to the luciferase (LUC) sequence except for the PTPRZ1-250 promoter, which stops at the ATG. The HRE consensus sequences and direction of each HRE are also indicated. The core HRE sequences are: HRE1, CCGTG; HRE2, CACGC; HRE3, CACGC; HRE4, CACGCACG; HRE5, CACGG.</p

Mutation of both LANA caspase cleavage sites leads to increased IL-1β production.

<p>THP-1 cells were matured overnight by treatment with TPA and then transiently transfected with FLAG-tagged forms of LANA-DM, WT-LANA, LANA-Nmut, or LANA-Cmut. The next day cells were treated with LPS and the level of IL-1β in the supernatants measured 20 hrs later. Cell extracts were also made for protein analysis and immunoblots. (A) IL-1β levels as determined by ELISA following transfection with the LANA plasmid constructs and treatment with LPS. The average level of IL-1β detected following transfection with vector alone (no LPS) was 2131 pg/ml (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005064#ppat.1005064.s003" target="_blank">S3 Fig</a>). (B) Immunoblot for cleaved caspase-1 and actin showing the relative levels of active casapse-1 compared to vector alone for the LPS-treated cells using the LiCor system. Data shown in (A) are the average +/- the standard deviation from 4 independent experiments. ** <i>P</i>< 0.01, *** P<0.005 for two-tailed Student’s t-test. Note that the average values for the single mutants were not statistically significantly different (p>0.05) when compared to wild type.</p

Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses

<div><p>Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of three hyperproliferative disorders: Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During viral latency a small subset of viral genes are produced, including KSHV latency-associated nuclear antigen (LANA), which help the virus thwart cellular defense responses. We found that exposure of KSHV-infected cells to oxidative stress, or other inducers of apoptosis and caspase activation, led to processing of LANA and that this processing could be inhibited with the pan-caspase inhibitor Z-VAD-FMK. Using sequence, peptide, and mutational analysis, two caspase cleavage sites within LANA were identified: a site for caspase-3 type caspases at the N-terminus and a site for caspase-1 and-3 type caspases at the C-terminus. Using LANA expression plasmids, we demonstrated that mutation of these cleavage sites prevents caspase-1 and caspase-3 processing of LANA. This indicates that these are the principal sites that are susceptible to caspase cleavage. Using peptides spanning the identified LANA cleavage sites, we show that caspase activity can be inhibited <i>in vitro</i> and that a cell-permeable peptide spanning the C-terminal cleavage site could inhibit cleavage of poly (ADP-ribose) polymerase and increase viability in cells undergoing etoposide-induced apoptosis. The C-terminal peptide of LANA also inhibited interleukin-1beta (IL-1β) production from lipopolysaccharide-treated THP-1 cells by more than 50%. Furthermore, mutation of the two cleavage sites in LANA led to a significant increase in IL-1β production in transfected THP-1 cells; this provides evidence that these sites function to blunt the inflammasome, which is known to be activated in latently infected PEL cells. These results suggest that specific caspase cleavage sites in KSHV LANA function to blunt apoptosis as well as interfere with the caspase-1-mediated inflammasome, thus thwarting key cellular defense mechanisms.</p></div

Changes in KSHV LANA following treatment of BCBL-1 cells with varying concentrations of H₂O₂ and sequence analysis of LANA identifying potential caspase cleavage sites.

<p>(A) BCBL-1 cells were cultured in media at 500,000 cells per ml to which was added H₂O₂ at 100 and 200 μM (diluted into PBS) or PBS as a control for 20 hrs. Nuclear and cytoplasmic extracts (10 μgs per sample) were prepared and analyzed by western blot for LANA using a monoclonal antibody that is directed towards the C-terminal region of LANA (Leica), and with antibodies directed toward TBP (rabbit) and HSP90 (mouse). Blots were then incubated with appropriate secondary antibodies (goat anti-mouse or rabbit IR800 secondary antibody) and analyzed using the LiCor system. Full length LANA (LANA-fl) and three faster migrating forms of LANA (arrows) are indicated. Molecular weight markers in kDa are indicated to the left of the blot. Full length LANA (calculated molecular weight of 126 kDa based on the amino acid sequence) migrates at approximately 165 kDa in the Nupage LDS gel system. HSP90 is shown as a cytoplasmic loading control and TBP as a nuclear loading control. (B) The 1095 LANA amino acid sequence translated from the KSHV genome derived from BCBL-1 cells (Genbank U93872) was analyzed for potential caspase cleavage sites utilizing the caspase webserver Cascleave (<a href="http://sunflower.kuicr.kyoto-u.ac.jp/~sjn/Cascleave/webserver.html" target="_blank">http://sunflower.kuicr.kyoto-u.ac.jp/~sjn/Cascleave/webserver.html</a>). Two sites with high probability scores were located within the N-terminal domain of LANA, and one was located in the C-terminal domain. Shown in bold and underlined are these three putative caspase cleavage sites; cleavage by caspases is predicted to occur at the carboxyl side of aspartate as indicated by an asterisk (*). (C) The two peptides that were found to be cleaved by caspase-1 and/or -3 are shown and denoted as LP-Nterm and LP-Cterm (with Cascleave scores of 0.980 and 0.840, respectively). (D) Reverse phase HPLC of 1mM LP-Nterm (MW 1919 Da) following treatment with PBS control (top panel), caspase-1 at 2.5 units/μl (middle panel), or caspase-3 at 0.25 units/μl (bottom panel). The new peak (bottom panel) represented the expected mass of 1182 Da for the N-terminal product if cleaved after the aspartic acid as shown (the C-terminal product GRECGPH was not detected under these assay conditions). (E) Reverse phase HPLC of 1mM LP-Cterm (MW 2040 Da) following treatment with vehicle control (top panel), caspase-1 at 2.5 units/μl (middle panel), or caspase-3 at 0.25 units/μl (bottom panel). The caspase-1 and 3 cleavage products identified by mass spectrometry are indicated in the middle and lower panels. The new peaks generated represented the expected masses of 1127 Da (10 minute peak) and 931 Da (12 minute peak) (F) Depiction of full length LANA and the five different forms (cp1-cp5) of BCBL-1 LANA expected to be generated following caspase cleavage at the 2 caspase cleavage sites identified from peptide analysis.</p

Caspase inhibitors block oxidative stress-induced changes in LANA.

<p>BCBL-1 (A, B) or BC-3 (C, D) cells were treated with PBS vehicle control or 100 μM H₂O₂ in the presence of 50 μM Z-FA-FMK (- ctrl), a negative control peptide (lanes 1 and 2), ZVAD-FMK (ZVAD), a pan-caspase inhibitor (lanes 3 and 4), a specific inhibitor of caspase-1 (C1 inh) (lanes 5 and 6) or a specific inhibitor of caspase-3/7 (C3/7 inh) (lanes 7 and 8). Nuclear (A, C) and cytoplasmic (B, D) extracts were harvested and LANA expression was analyzed by western blot and probed with antibody to FLAG, anti-rabbit secondary antibody conjugated to alkaline phosphatase and visualized using stabilized Western Blue substrate (Promega). Results are representative of 3 separate experiments for controls and ZVAD and 2 separate experiments for the C1 and C3/7 inhibitors. Molecular weight markers are shown to the left. To the right of the blots the different forms of LANA are indicated and include the location for full length LANA (LANA-fl) and three forms of LANA migrating below full length LANA with presumptive designations as LANAcp3, LANAcp2 and LANAcp5 based on their mobility.</p

Peptides containing LANA caspase cleavage sites inhibit IL-1β production.

<p>Cell permeable peptides with an N-terminal cellular HIV-1 Tat delivery sequence RKKRRQRRR containing the N-terminal caspase cleavage site of LANA (T-LP-Nterm) or the C-terminal caspase cleavage site of LANA (T-LP-Cterm) were tested as potential inhibitors of IL-1β production following treatment of THP-1 cells with LPS. ZVAD was used as a positive caspase inhibitor control. (A) Effect of ZVAD (50 μM), T-LP-Nterm (50 μM) and T-LP-Cterm (50 μM) on IL-1β in the supernatant from THP-1-treated cells. Δata is from five separate experiments (except for T-LP-Nterm, 4 experiments). ***, <i>P</i>< 0.0005, **** <i>P</i>< 0.0001 for two tailed paired Student’s t-test. The average IL-1β λεωελ ϕορ untreated and LPS treated THP-1 cells was 276 pg/ml and 2344 pg/ml, respectively. (B) Immunoblot analysis for cleaved (mature) caspase-1 and actin as a loading control. The fold change in cleaved caspase expression normalized to β-actin is shown just below the immunoblot using the LiCor system. (C) Immunoblot analysis for Pro-IL-1β and actin as a loading control. The fold change in Pro-IL-1β expression normalized to β-actin is shown just below the immunoblot using the LiCor system.</p