Scholarly Activity Improvement Fund (SAIF)-A 2012/2013 Final Report. Blocking signal pathway inter-connectors to reverse cell death

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1 Scholarly Activity Improvement Fund (SAIF)-A 2012/2013 Final Report Blocking signal pathway inter-connectors to reverse cell death Chanaka Mendis Professor of Chemistry Department of Chemistry & Engineering Physics University of Wisconsin Platteville Platteville, WI 53818

2 Title; Blocking signal pathway inter-connectors to reverse cell death Introduction; Signaling proteins coordinate various cellular processes in response to extracellular stimulants operating within obscure networks of thousands of different regulatory proteins. These signal transduction networks link receptors to extracellular mediators at the cell surface and react appropriately to benefit the organism, real havoc ensues when these proteins malfunction as a consequence of mutations in the genes that encode them. A common obstacle that has been hampering the efforts of targeting components that are initially effective as successful signal blockers in disease control is the inability of the drug to be effective over an extended time period. SEB, which is known to impinge on cellular pathways including pathways controlling apoptosis [1] also induce symptoms such as vomiting, diarrhea, vertigo and muscle weakness. Following 24 hours of exposure, these symptoms may develop further to induce hypotension and vasodilation of blood vessels in kidneys and other organs [2]. Lipopolysaccharide (LPS), an endotoxin frequently seen in the cell wall of Gram-negative organisms, stimulates various immune responses including secretion of cytokines and activation of macrophages and lymphocytes [2]. Previous work done in our laboratory did not only identify multiple signaling pathways induced in a SEB/PBMC and a LPS/PBMC modules but also crosstalk between these pathway inter-connectors. Inhibiting pathway inter-connectors and identifying multiple signaling activities in SEB induced PBMC s have significantly reduced the so called leaking effect. We have identified two such targets (p38 and JNK) once inhibited showed reduced activity upon toxin induction [3]. The purpose of this study was to further investigate human peripheral blood mononuclear cells (PBMC) in order to better understand the apoptosis related events and strategies to reverse such events induced by SEB and LPS. C-Jun N-Terminal Kinase (JNK) and

3 p38 have been previously identified to be induced by both SEB and LPS as well as to interconnect multiple signaling cascades. Our goal was to better understand the effect of these two pathway inter-connectors (JNK and p38) in these two experimental models by targeting and inhibiting then selectively and study the effect on cell death. Previous work done in our laboratory has also identified a set of genetic and cellular markers specific for septic shock inducing LPS in human PBMCs as well as for cell death [Unpublished results]. We used these genetic and cellular markers as a baseline to further nvestigate the influence of the key pathwayinter-connectors utilizing a dual expression front. Methods QUANTIFICATION OF RNA AND PROTEIN SAMPLES: RNA and protein samples were isolated from three types of human PBMCs (Peripheral Blood Mononuclear Cells): 1) control (not treated with anything at 2 and 6 hours), 2) toxin exposed cells with inhibitor for 2 and 6 hrs, and 3) toxin exposed cells without the inhibitor at 2 and 6 hours (WRAIR, Silver Spring, MD). These samples were then quantitated using a UV-VIS spectrometer (PerkinElmer Lambda 900 UV/VIS/NIR Spectrometer). REVERSE TRANSCRIPTION (RT-PCR);RT was done using an iscript cdna Synthesis Kit (BIO-RAD) in a Thermocycler (Eppendorf Mastercyler Personal). POLYMERASE CHAIN REACTION (PCR);PCR was carried out using primers of genes of interest designed through various kinds of primer-design software in a Thermocycler (Eppendorf Mastercycler Personal) using a PCR master mix kit (Roche Diagnostics, Indianapolis, IN) VISUALIZATION & QUANTIFICATION; mall PCR samples were analyzed on 1% agarose gels using syber green for fluorescence and were visualized through an in house imaging apparatus and quantitated by Image J analysis.

4 ELISA; All samples were either run using the Caspase-3, Caspase-8, or Caspase-10 Colorimetric Assay Kits from BioVision (Mountain View, CA). The type of instrument used was a Microplate Autoreader EL311 Bio-Tek Instrument. Results and Discussion Previous research has discovered that SOD genes regulate caspase-1 activation through reversible oxidation and glutathionylation of cysteine residues (Meissner). It has also been discovered that USP has been shown to play a large part in caspase regulation and cell survival through multiple ubiquitin pathway proteins and their effect on targeted degradation via the ubiquitin-proteasome pathway, specifically with its effect on the gene IAP (Steller). With this knowledge, a set of genes was selected in order to attempt to piece a signaling pathway together. From the results obtained thus far, Caspase 1, 6, 7, 9, 10, HEP, SOD, USP, STK17A, and REQ were all up-regulated at both the 2 and 6 hour exposures by SEB (Table 1). The results also showed that the p38 inhibitor SB was able to alter the expression of all SEB induced apoptosis related genes (Table 1). From these results, it is clear that SEB affects the intrinsic apoptosis pathway. The intrinsic apoptosis pathway consists of the induction of Caspase 9 followed by the induction of Caspase 3, 6, and 7. On the other hand, the extrinsic apoptosis pathway consists of the induction of Caspase 8 followed by the induction of Caspase 10, 3, 6, and 7. Due to the values of 16.9 ± 2.0 (SEB 2 hr) and 10.4 ± 0.4 (SEB 6 hr) it can be said that SEB affects the apoptosis pathway due to the up-regulation of Caspase 9. In conjunction with the testing of RNA samples with the genes of interest, protein testing has begun with Caspase 3 and Caspase 8 (Table 2). As done with the RNA data, calculations are performed to normalize the absorbance readings and from this data no conclusive results as to

5 the apoptosis pathway affected can be obtained due to the fact that the protein of Caspase 9 has not been tested yet. Gene Time Counts 2 hr Time Counts 6 hr 24 hr p38 (inhibitor) Caspase ± ± ± 0.4 Caspase ± ± ± 0.5 Caspase ± ± ± 0.6 Caspase ± ± ± 3.3 Caspase ± ± ± 0.4 HEP 11.2 ± ± ± 0.1 SOD 7.0 ± ± ± 0.1 USP 8.1 ± ± ± 0.3 STK17A 13.0 ± ± ± 0.2 REQ 4.8 ± ± ± 0.2 Table 1: These calculated regulation values were obtained using the absorbance value of the gene of interest and the housekeeping gene. Gene Sample Absorbance at 405 nm Calculated Values: #/C Caspase 3 SEB-SP 2H A Caspase 3 5-LPS SP A Caspase 3 SEB 12 H B Caspase 3 5-LPS PD B Caspase 3 LPS 2H B Caspase 3 SEB-SB 2H A Caspase 3 LPS 2-C A Caspase 8 SEB-SP 2H A

6 Caspase 8 5-LPS SP A Caspase 8 SEB 12 H B Caspase 8 5-LPS PD B Caspase 8 LPS 2H B Caspase 8 SEB-SB 2H A Caspase 8 LPS 2-C A Table 2: Elisa absorbance readings which were prepared using Caspase 3 and 8 kits. Conclusion and Future Plans While there are still many genes to be tested in potential apoptosis pathways of human peripheral blood mononuclear cells, this project yielded promising results. Since a lot of the genes tested have been effectively down-regulated by the p38 pathway inhibitor SB203580, the potential for mapping out the apoptotic pathway of the SEB toxin is growing with each additional gene studied. Currently, reproducible results are in the process of being obtained with other pathway inter-connectors and with LPS treated samples as well. This will allow us to confirm this observation, along with other genes that are associated with apoptosis: GRIM 19, NGFRAP1, MAPKAPK5, PDCD4, MIHC, Caspase 3, and Caspase 8. As the data pool grows, the apoptotic pathway can be traced and confirmed, thus being a vital study to the hopes of inhibiting cell death in both LPS and SEB induced human PBMCs. References Blazes, David L., Lawler, James V., Lazarus, & Angeline A. When Biotoxins are Tools of Terror. Postgraduate Medicine, Vol. 112:2. Section Symposium: Third of Three Articles on Bioterrorism Jett, Marti, Ionin, B., Das, R., & Neill, R. The Staphylococcal Enterotoxins. Walter Reed Army Institute of Research, Silver Spring, MD, USA Meissner, Felix, K. Molawi, A Zychlinsky. Superoxide dismutase 1 regulates caspase-1 and endotoxic shock Nature Immunology, Vol. 9:8 pp: August 2008.

7 Mendis, Chanaka, Campbell, K., Das, R., Yang D., & Jett, M. Effect of 5-Lipoxygenase inhibitor MK591 on early molecular and signaling events induced by staphylococcal enterotoxin B (SEB) in human peripheral blood mononuclear cells (PBMCs) FEBS Journal, May 2008 Steller, H. Regulation of apoptosis in Drosphilia Cell Death and Differentiation, (15)