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Regulation of ORMDL3 Gene Expression through the ERK/CREB Pathway Annie Vo.

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Presentation on theme: "Regulation of ORMDL3 Gene Expression through the ERK/CREB Pathway Annie Vo."— Presentation transcript:

1 Regulation of ORMDL3 Gene Expression through the ERK/CREB Pathway Annie Vo

2 Asthma Chronic inflammatory disease Airway inflammation Airway hyperreactivity Increasing every year, particularly in developed countries 5-16% of the world population afflicted Figure 1 Asthma in the U.S from 2001 to 2011. A slight increase in asthma prevalence in various groups evidence in recent years. Children are consistently the biggest population in the total count.

3 Vitamin A Supplementation Lowered amounts found in asthmatic patients Vitamin A involved with enhancing maintenance of airways Instead, increased inflammatory factors Mechanism not fully understood Zhuang et al. (2013) proposed a mechanism by which vitamin A increased the onset of asthma.

4 How does vitamin A affect asthma? Vitamin A

5 How does vitamin A affect asthma? Vitamin AATRA (all-trans retinoic acid)

6 How does vitamin A affect asthma? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA* (activated)

7 How does vitamin A affect asthma? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated)

8 How does vitamin A affect asthma? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene

9 How does vitamin A affect asthma? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

10 What is ORMDL3? Orosomucoid 1-like 3 Associated with onset of childhood asthma Expressed in bronchial epithelial cells Encodes an ER trans-membrane protein Binds to the Ca 2+ pump on the sacro-endoplasmic reticulum Imbalance of Ca 2+ increase the unfolded protein response in ER Causes upregulation of inflammatory factors

11 Testing the model: Does ATRA increase P-CREB? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

12 Testing the model: Does ATRA increase P-CREB? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

13 Gel Electrophoresis

14 Electroblotting

15 Western Blot

16 Does ATRA increase P-CREB? (Zhuang et al. 2013)

17 Does ATRA increase ORMDL3 expression? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

18 Does ATRA increase ORMDL3 expression? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

19 Does ATRA increase ORMDL3 expression? (Zhuang et al. 2013) Is ATRA-induced ORMDL3 expression dependent on PKA?

20 Is expression dependent on PKA? Vitamin AATRA (all-trans retinoic acid) PKA (protein kinase A) PKA*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3 H-89

21 Is expression dependent on PKA? (Zhuang et al. 2013)

22 Are there other kinases that lead to ORMDL3 expression? Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

23 Are there other kinases that lead to ORMDL3 expression? Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3

24 Are there other kinases that lead to ORMDL3 expression? Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3 ? ?

25 Methods Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3PKA H-89

26 Methods Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3 ? ? PKA H-89

27 Methods Vitamin AATRA (all-trans retinoic acid) ERK1/2 (Extracellular signal- regulated kinase-1 and 2 ) ERK1/2*CREB (inactive transcription factor) (activated) P-CREB (phosphorylated) ORMDL3 gene ORMDL3 ? ? PKA H-89

28 Methods Controlling kinases Inhibition of ERK1/2 using U0126 Inhibition of PKA using H-89 Expression of ORMDL3 Western Blot Near-infrared (NIR) secondary antibody Measurement of luminescence to determine protein concentration

29 Expected Results ORMDL3 will be when either PKA or ERK1/2 is present. A kinase must be present for ORMDL3 to be expressed.

30 Expected Results ORMDL3 will be when either PKA or ERK1/2 is present. A kinase must be present for ORMDL3 to be expressed.

31 Future Uses Application: Regulation of vitamin A intake through diet during childhood years to prevent asthma. Future Questions: Synergistic response when both are present? Why is the amount of protein produced different when varying kinases?

32 References Allen, S., Britton, J. R., & Leonardi-Bee, J. a. (2009). Association between antioxidant vitamins and asthma outcome measures: systematic review and meta-analysis. Thorax, 64(7), 610–9. Dijk, F. N., de Jongste, J. C., Postma, D. S., & Koppelman, G. H. (2013). Genetics of onset of asthma. Current opinion in allergy and clinical immunology, 13(2), 193–202. GeneLoc. "ORMDL3 Gene in genomic location." GeneCard. Accessed December 5, 2013. http://www.genecards.org/cgi-bin/carddisp.pl?gene=ORMDL3. Ha, S. G., Ge, X. N., Bahaie, N. S., Kang, B. N., Rao, A., Rao, S. P., & Sriramarao, P. (2013). ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48. Nature communications, 4, 2479. Kang, M.-J., Yu, H.-S., Seo, J.-H., Kim, H.-Y., Jung, Y.-H., Kim, Y.-J., … Hong, S.-J. (2012). GSDMB/ORMDL3 variants contribute to asthma susceptibility and eosinophil-mediated bronchial hyperresponsiveness. Human immunology, 73(9), 954–9. Leinco. “Detection in Western Blots." Leinco. Accessed December 5, 2013. http://www.leinco.com/general_wb. Leinco. "Western Blot Setup." Leinco. Accessed December 5, 2013. http://www.leinco.com/general_wb. Martinez, F. D., & Vercelli, D. (2013). Asthma. Lancet, 382(9901), 1360–72. National Institute of Allergy and Infectious Diseases, National Institutes of Health. Asthma in the U.S. (http://www.niaid.nih.gov/topics/asthma/understanding/Pages/usAsthma.aspx; Accessed December 2013) The Vitamin Magazine. "Great Sources of Vitamin A." The Vitamin Mag. Accessed December 5, 2013. http://www.thevitaminmag.com/vitamin-a-foods-foods-high-in-vitamin-a/. Zhuang, L.-L., Huang, B.-X., Feng, J., Zhu, L.-H., Jin, R., Qiu, L.-Z., & Zhou, G.-P. (2013). All-Trans Retinoic Acid Modulates ORMDL3 Expression via Transcriptional Regulation. PloS one, 8(10), e77304. Zhuang, L.-L., Jin, R., Zhu, L.-H., Xu, H.-G., Li, Y., Gao, S., … Zhou, G.-P. (2013). Promoter characterization and role of cAMP/PKA/CREB in the basal transcription of the mouse ORMDL3 gene. PloS one, 8(4), e60630.

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