1. Cell Signaling, Wound Repair, and ATP Receptors Kevin Quirke and Alex Knobloch

2. Cell Signaling Overview typical cell exposed to numbers of different signal molecules –selective response according to cell function –cellular response dictated by: unique sets of cell surface receptors cell-specific intracellular targets

3. Cell Signaling Overview Essential Cell Biology, 2 nd Edition (Alberts et al., 2004) altered metabolism altered gene expression altered cell shape or movement expression

4. Examples of Cell Signals growth/mitotic signaling: epidermal growth factor (EFG) EFG-receptor (EGFR) Essential Cell Biology, 2 nd Edition (Alberts et al., 2004)

5. Examples of Cell Signals growth/mitotic signaling: activated EFG- receptor (EGFR) EFG Essential Cell Biology, 2 nd Edition (Alberts et al., 2004)

6. Examples of Cell Signals growth/mitotic signaling: active Ras protein activation of transcription factors for pro-mitotic genes phosphorylation cascade Essential Cell Biology, 2 nd Edition (Alberts et al., 2004)

7. Process Where Cell Signaling is Important = Wound Repair

8. Wound Repair 1.Inflammatory 2.Proliferative 3.Remodeling Tissue repair following injury Three phases 6 :

9. Examples of Signals in Inflammatory Phase PAMPs DAMPs

10. PAMPs Pathogen-associated molecular patterns (PAMPs) Microbial molecules targeted by macrophages Teichoic acid Bacterial Cell

11. Innate Immune Response to PAMPs macrophage bacterium PAMP Receptor cytokines Inflammation

12. DAMPs Damage-associated molecular-patterns (DAMPs) Intracellular molecules Released by cells undergoing stress or death Initiate immune response

13. Extracellular DNA as a DAMP Extracellular Space

14. Receptor Extracellular DNA as a DAMP Extracellular Space macrophage cytokines Inflammation

15. Key Recipient and Producer of Inflammatory Phase Signals = Monocytes

16. Monocyte White blood cell Produced in boned marrow Differentiates in tissues Participate in wound repair Bone Marrow Stem Cell Blood Monocyte Tissue Macrophage Phagocytes Abbas et al., 2009 University of New England

17. Monocyte’s Role in Wound Repair 1.Inflammatory 2.Proliferative 3.Remodeling Three phases:

18. Monocyte’s Role in Wound Repair 1.Produce inflammatory mediators 2.Produce pro-angiogenic factors 3.Phagocytose cellular debris monocyte cytokines monocyte Pro-angiogenic factors monocyte

19. VEGF Vascular endothelial growth factor (VEGF) Chemical signal Pro-angiogenic factor Promotes wound repair

20. VEGF’s Role in Wound Repair 1.Inflammatory 2.Proliferative 3.Remodeling Three phases:

21. VEGF’s Role in Wound Repair Endothelial cell 1.New blood vessel formation (angiogenesis) 2.Endothelial cell proliferation 3.Oxygen supply to tissues Promotes: www.evgn.org

22. VEGF Triggers of VEGF production: 1.Hypoxia 2.Oncogenes 3.Other growth factors and cytokines 4.Cellular receptors

23. VEGF VEGF receptor VEGF monocyte Blood Vessel Cell signal Trigger: Cellular receptor Target: Endothelial cell VEGF Receptor

24. ATP = one signal molecule to which monocytes respond

25. ATP as an Extracellular Signal roles as signal molecule: –DAMP inflammatory response 1, pain sensation 2 –synaptic signaling (neurotransmitter) 2,3 –neuron-glia signaling 4 –muscle contraction 5 adenosine-5'-triphosphate (ATP)

26. P2 Nucleotide Receptors family of nucleotide cell surface receptors two major subfamilies P2Y Receptors - G-protein coupled 1 - bind ATP, ADP, UTP, and UDP 1 Essential Cell Biology, 2 nd Edition (Alberts et al., 2004)

27. P2 Nucleotide Receptors family of nucleotide cell surface receptors two major subfamilies P2Y Receptors - G-protein coupled 1 - bind ATP, ADP, UTP, and UDP 1 P2X Receptors - ionotrophic (ligand-gated) 1 - bind ATP 1 wikipedia.org

28. P2X Subfamily ligand-gated ion channels cation selective 2 –equal permeability to K + and Na + –significant permeability to Ca 2+ seven members (P2X 1-7 )

29. P2X 7 aka P2RX7

30. P2X 7 aka P2RX7 Zebrafish P2X4 (Kawate et al., 2009)

31. P2X 7 aka P2RX7 wikipedia.org

32. P2X 7 ligand-gated ion channel Essential Cell Biology, 2 nd Edition (Alberts et al., 2004) ATP

33. P2X 7 requires high concentrations (mM) of ATP for activation 1 sensitive to synthetic ATP analog BzATP 1 adenosine-5'-triphosphate (ATP) 3′-0-(4-benzoyl) benzoyl ATP (BzATP)

34. P2X 7 requires high concentrations (mM) of ATP for activation 1 sensitive to synthetic ATP analog BzATP 1 adenosine-5'-triphosphate (ATP) 3′-0-(4-benzoyl) benzoyl ATP (BzATP)

35. P2X 7 major immune modulator activation in monocytes IL-1β nitric oxide synthase ROS production of pro-inflammatory molecules (Lenertz et al., 2009)

36. The Hill et al. paper suggests a role for ATP and P2X 7 in the proliferative phase of wound repair –Counterintuitive considering its role in the inflammatory phase…

37. ATP VEGF Cell P2RX7 Angiogenesis monocyte

38. References 1. Lenertz, L.Y., M.L. Gavala, L.M. Hill, and P.J. Bertics. 2009. Cell signaling via the P2X 7 nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking. Purinergic Signal 5: 175-187. 2. Khakh, B.S. 2001. Molecular physiology of P2X receptors and ATP signalling at synapses. Nat. Rev. Neurosci. 2: 165-174. 3. Khakh, B.S., and R.A North. 2006. P2X receptors as cell-surface ATP sensors in health and disease. Nature 442: 527-532. 4. Fields, R.D., and G. Burnstock. 2006. Purinergic signalling in neuron-glia interactions. Nat. Rev. Neurosci. 7: 423-436. 5. Vassort, G. 2001. Adenosine 5′-triphosphate: a P2-purinergic agonist in the myocardium. Physiol. Rev. 81: 767-806. 6. Kirsner, R.S., and W.H. Eaglstein. 1993. The wound healing process. Dermatol. Clin. 11: 629-640. 7. Abbas, A.K., and A.H. Lichtman. 2009. Basic immunology: functions and disorders of the immune system. Saunders: Philadelphia, pp. 24-29.