Extracellular PKM 2 Promotes Wound Healing ZhiRen Liu

Extracellular PKM 2 – Promotes Wound Healing Zhi-Ren Liu Department of Biology, GSU

Research Directions Functional roles of p 68 RNA helicase in cancer progression and metastasis Tumor bio-energetic, role of PKM 2 in cancer progression Extracellular PKM 2 in tissue regeneration Development of Diagnostic and therapeutic agents by protein engineering (Joint projects with Jenny Yang) Page 2

PKM 2 in Glucose Metabolism in Cancer Cells Pentose phosphate pathway NADPH+ribose 5 -Pi PKM 2 Warburg Effect Cancer cells use glycolysis even in the presence of oxygen Page 3

PKM 2 Is a Protein Kinase in Gene Expressions Glucose G-6 -PPentose Phosphate Pathway NADPH, R 5 P Glycolysis Growth signals PEP PKM 2 active Pyruvate inactive P Stat 3 RNA Pol II MEK 5 Gao, X, et. al. Mol. Cel. , 2012 Page 4

Extracellular PKM 2, A Diagnostic/Prognostic Marker PKM 2 levels in blood circulation or stool are diagnosis/prognosis marker How Does PKM 2 released to blood circulation? Does circulative PKM 2 have any functional role in cancer progression? Thousands Refs Page 5

Extracellular PKM 2 Promotes Tumor Angiogenesis Glucose G-6 -PPentose Phosphate Pathway NADPH, R 5 P Glycolysis Growth signals PEP PKM 2 active Pyruvate Stroma Cells inactive Endothelial Cells Cancer Cells Angiogenesis, Metastasis, & drug resistance Page 6 P Stat 3 RNA Pol II MEK 5

Wound Healing Processes 1. Hemostasis 2. Inflammation. 3. Proliferation 4. Maturation Page 7

Topical Application of r. PKM 2 Facilitates Wound Healing Page 8

Extracellular r. PKM 2 Promotes Angiogenesis at Wound Site Green: CD 31 Stains Page 9

Age Effects Mouse Age 9 -10 weeks 90 % of healing 80 r. PKM 2 70 r. PKM 1 60 Buffer Mouse Age 5 weeks 100 80 60 50 40 Pro. Woud r. PKM 2 Control r. PKM 1 Buffer 40 30 20 20 10 0 Page 10 Day 3 Day 6 Day 9

PKM 2 Neutralize Antibody Delayed Wound Healing Page 11

Extracellular PKM 2 is released to the wound site by an intrinsic mechanism IHC via a-PKM 2 Page 12

PKM 2 Released by Nutrophils at Wound Site PKM 2 Neutrophils No wound Page 13 Day 1 Day 2 Day 3 Day 4

PKM 2 Released by Nutrophils at Wound Site Neutrophils isolated from mouse blood Dam is activator for primary and 2 nd neutrophil degranulation Page 14 IHC of wound tissue sections Beige-J mice have defects in neutrophil migration

r. PKM 2 Promotes Granulation at Wound Buffer The PKM 2 treatment group had much better wound healing. Granulations were clearly started early time. There were also obvious richer in vessels in the wound areas in the PKM 2 treatment group. r. PKM 1 Granulations r. PKM 2 Page 15

Extracellular PKM 2 Promotes Fibroblast Cell Migration Green: Fibroblast Blue: DAPI HDFa Cells: Primary human dermal fibroblast cells Page 16

Extracellular PKM 2 promotes myofibroblast differentiation Sections from wound tissue PKM 1 PKM 2 Green: a-SMA Blue: DAPI TGF-β HDFa cells Page 17 Red: a-SMA Blue: DAPI

r. PKM 2 Promotes Wound Healing with Diabetic Mice Diabetic db/db mice at 15 weeks old r. PKM 2 and r. PKM 1 were used in 0. 04% W/W in pharmacy cream and water in PBS % of healing 100 r. PKM 2 80 day 0 Buffer 60 r. PKM 1 day 3 day 6 day 9 r. PKM 1 40 r. PKM 2 20 0 Day 3 Day 6 Day 9 % of healing = Initial wound areas - measured wound areas at a given day/initial wound areas The error bars are the standard deviations among the five mice. Page 18

Acknowledgments Collaborators: Dr. Jenny J. Yang Dept. of Chemistry Georgia State Univ. Dr. Shi-Yong Sun Winship Cancer Institute Emory University Dr. Z. G. Chen Winship Cancer Institute Emory University Dr. Xiao-Ping Hu Dr. Hui Mao Members of Dr. Yang’s laboratory, Dept. of Chem, GSU • National Institute of Health Ms. Birgit Neuhaus Dept. of Biology GSU. Dr. Shiming Wang Dept. of Chemistry GSU. Page 19 • American Heart Association • Georgia Cancer Coalition • MBD pre-doctoral fellowship. Emory University

Extracellular PKM 2 Interacts with Integrins avb 3 av b 3 Page 20