Novel Physiological Role of Caveolin-1 in Aging and Aging-related Diseases. Sang Chul Park Gachon University Lee Gil Ya Cancer and Diabetes Institute

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1 Novel Physiological Role of Caveolin-1 in Aging and Aging-related Diseases Sang Chul Park Gachon University Lee Gil Ya Cancer and Diabetes Institute

2 Primarily I asked questions on biological issues on aging Parenchymal loss Functional loss Morphological alteration

3 Question 1 Apoptosis for parenchymal loss

4 Young UV Old DNA Fragmentation by UV-irradiation UV-Induced Activation of SAPK/JNK UV Young M Old M (J/m 2 ) UV (J / m 2 ) Young Old

5 Suh et al.naturemedicine,2002,8,3 Young MMS? Old.. Absence of Apoptotic Response to MMS in vivo 0 Hrs 2 Hrs Young Old No.of apoptotic cells/ 50,000 cells young old Time (Hrs) after MMS tx.

6 DAPI p-p38 MERGE O +Stau Control Y +Stau Control A 노화에따른세포사멸신호핵내이동차단 B DAPI p-jnk MERGE 2+ H 2O O Control 2+ H 2O Y Control Tx : stau 50nM for 60 min Tx : H 2 O 2 100uM for 60 min

7 Nuclear Barrier Hypothesis of Aging Membrane Membrane Cytosol Cytosol Nucleus Nucleus

8 노화현상의 비가역성과불가피성의 생물학적근거 텔로미어단축 노화색소축적 생체분자의손상

9 C D Y O O R Y O O R Hela O on Y-ECM /5.0/ /3.5 htert (183bp) Hela Y O (day) ITAS GAPDH

10 DNA damage repair in restoration A Y O O R B Y TTAGGG-FISH ƔH2AX Merge Enlarged O Tail moment Tail moment ** Y O O R O R TIF positive cells (%) ** Y O O R

11 노화현상의 비가역성과불가피성의 생물학적근거 : 부정되고있음 텔로미어단축 : 제어가능 노화색소축적 : 제어가능 생체분자의손상 : 제어가능

12 Aging phenotype is an adaptive response for Survival toward environmental stress through resistance to apoptotic stress Possibility of Restore Principle Instead of Replace Principle

13 Question 2 Loss of Functional Efficiency

14 Senescence-associated cellular responses 1. Metabolic response stability essential 2. Stress response unstable response 3. Mitogenic response No response or low response Major source of irreversibility nature of aging

15 Aging effect on Caveolin expression Cav-1 Cortex Mid Br Hippo Cbll Heart Lung Liver Kidney Spleen Y O Y O Y O Y O Y O Y O Y O Y O Y O Cav-2 Coomassie stain Park et al JBC 2000

16 Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. Park, et al. (2000 ) J Biol Chem. 275, Park et al(2001) Ann NYAcad Sc, 2001,928,79 In young cells EGF Park et al(2002)ann NYAcad Sc, 2002,959,45 EGF In old cells EGFR -P EGFR Caveolin-1 ` Aging ` Erk-1/2 -P Erk-1/2

17 Senescent phenotype can be reversed by reduction of caveolin status In old cells Cho et al. (2003) J Biol Chem. 278(30):27789 Park (2002) Mech.Age.Dev, 123,917 EGF In old cells EGFR EGF Caveolin-1 - caveolin EGFR -P Erk-1/2 -P Erk-1/2 p53 Elk -P p21 Cell cycle progression

18 Question 3 Morphological alteration

19 ECM Integrin Cav-1 Rac P- FAK Pax -P Cdc42 F-actin Disruption of focal adhesion by down-regulation of caveolin?

20 Morphological adjustment of senescent cells sirna of cav-1 O-GL O-Cav O-GL FAK p-fak Cav-1 Actin O-CAV Cho et al., J Biol Chem. 2004; 279(40):42270 Focal adhesion Actin stress fiber Nucleus

21 Morphological adjustment of the senescent cells can be induced by simple modulation of the caveolin status Structural restoration of the senescent cells by crosstalk between determinants of function and morphology

22 Caveolin in signal regulation, intracellular trafficking, polarity, migration, endocytosis and More!!

23 Question 4 Organ pathology level Diabetes Stem cell differentiation Cancer cell differentiation, Wound healing Exercise

24 Caveolin-1 and Aging-related Diseases Cancer Caveolin-1 as a negative regulator of the Ras-p42/44 MAP Kinase Cascade Alzheimer disease APP in caveolae Cav-1 promote secretase cleavage Caveolin-1 Diabetes Key roles in activation of insulin RTK and GLUT4 translocation Vascular disease Direct interaction with e-nos Activity inhibition of e-nos

25 Prevalence of Diabetes

26 Control of blood glucose levels Type 1 Diabetes Type 2 Diabetes β Cell Insulin x Liver Glucose Muscle Glucose α Cell Glucagon Fat Glucose Digestive System

27 Type II Diabetes - NIDDM (non-insulin dependent) 90-95% of DM Obese type and Lean type (65 %) - Insulin resistance - Mainly occurs in adults Is Ageing itself related with Type II DM? Why no adequate Age dependent lean type DM model?

28 Type II D.M Animal models GK OLETF ob/ob ZDF Ageing independent obese Few studies on obesity independent or age dependent lean type D.M

29 Roles of Caveolin-1 in Muscle Tissues?

30 Caveolae Small vesicular organelles of cell membrane Signaling molecules enriched in caveolae; G-protein subunits, EGFR and related receptor tyrosine kinases Caveolin, marker protein of caveolae Ubiquitous Cav-1 N TM C Cav-2 Muscle specific Cav-3

31 Caveolin & insulin sensitivity in adipocytes Relationship of caveolin with insulin signaling Insulin Glucose IR TC10 P Caveolin-1 IRS-1/2 GLUT-4 Erk-1/2 P PI3-K P Akt P

32 Can caveolins regulate the insulin signaling in skeletal muscle cells as in adipocytes?

33 Expression of caveolins during differentiation A Pan Liv Adi Mus Lu He Br Tes B Undi Di Cav-1 Cav-1 Cav-2 Cav-2 Cav-3 Cav-3 Tubulin IR Akt C Undifferentiated Differentiated GLUT4 Myogenin -actin D Insulin (-) Insulin (+)

34 Caveolins and GLUT-4 A Cav-1 GLUT-4 Nuclei Merged B Top Bottom Cav-1 - insulin - insulin Cav-3 GLUT-4 + insulin Top Bottom Cav-1 + insulin Cav-3 GLUT-4

35 Role of caveolins in glucose uptake A B Insulin (-) Insulin (+) Insulin (-) Insulin (+) GL sirna DeGlu uptake (relative basal) Cav-1 sirna Cav-1 sirna 0.5 sirna: 0 GL C1 C3 Cav-3 sirna Cav-1 Cav-3

36 Caveolin-1 in Muscle Aging?

37 Aging Effect on Caveolin-1 Status in Muscle and Fat Tissues Muscle Fat Lung Insulin (μiu/ml) Y O Y O Y O IR 20 GLUT-4 Cav Cav-3 0 Y O Common nature of muscle and fat tissue would be the insulin-sensitivity

38 NEW ASSUMPTION AGE DEPENDENT LOSS OF CAVEOLIN-1 IN MUSCLE AND FAT TISSUE WOULD BE CAUSATIVE FOR NON-OBESE TYPE II DIABETES? Caveolin-1 and Diabetes type II

39 JYD animal model Collaboration with University of Calgary (Dr. Ji-Won Yoon & Hee-Sook Jun) F M DBA X C57BL/6 Age dependent phenotype Sex dependent diabetic phenotype

40 Blood glucose levels, incidence of diabetes, and body weight in male JYD mice A B C Blood glucose (mg/dl) 300 JYD B6 250 DBA * ** ** ** Incidence of diabetes (%) JYD B6 DBA Body weight (g) JYD B6 DBA ** ** Age (wks) Age (wks) Age (wks)

41 Glucose and insulin tolerance tests in male JYD mice 10 Blood glucose (mg/dl) JYD B6 DBA * * * * weeks 30 Time (min) Time (min) Blood glucose (mg/dl) 600 JYD B6 500 DBA * * * weeks % of initial blood glucose JYD B6 DBA * * * % of initial blood glucose JYD B6 DBA * * * * Time (min) Time (min)

42 Whole-body 18 F-FDG PET images in JYD mice C57BL/6 ob/ob JYD M %ID/g 4.0 Harderian gland Brain Brown fat Heart Paraspinal muscle lung Liver Bowel Bladder 0.0

43 Expression of caveolins in skeletal muscle and other insulin responsive tissues A B Mus Adi Pan DBA/2 C57BL/6 JYD (wks) Cav-1 Cav-3 Tubulin Cav-1 Tubulin DBA/2 B6 JYD Can Caveolins regulate the insulin resistance in JYD model? Cav-1 C Tubulin Fold change * * D-M ND-F ND-M 3 wks 10 wks 30 wks * Liv Cav-1 Tubulin Cav-1 Cav-3 tubulin

44 Down-regulation of caveolin-1 by shlenti-cav-1 viral vector A shlenti-gfp ; CMV GFP U6 loop shlenti-cav-1 ; CMV GFP U6 Cav-1 B C Cav-1 actin shlenti-gfp shlenti-cav-1 shlenti-gfp shlenti-cav-1 D E shlenti-gfp shlenti-cav-1 Cav-1 Flotillin Cav-1 Flotillin shlenti-gfp shlenti-cav-1

45 Impairment of glucose and insulin tolerance in shlenti-cav-1-injected C57BL/6 mice A B Blood glucose (mg/dl) shlenti-gfp shlenti-cav-1 * * * Body weight (g) shlenti-gfp shlenti-cav-1 C (Day) D (weeks) Blood glucose (mg/dl) * shlenti-gfp shlenti-cav-1 % of initial blood glucose * * shlenti-gfp shlenti-cav Time (min) Time (min)

46 Overexpression of caveolin-1 by adenovirus-cav-1 viral vector A Ade-GFP ; CMV CMV EGFP Ade-cav-1 ; CMV Cav-1 CMV EGFP B C Cav-1 actin Ade-GFP Ade-cav-1 Ade-GFP Ade-cav-1-GFP D E Mus CON 3 DAYS IV IM GFP Ade-GFP Ade-cav-1 Cav-1 Flotillin Cav-1 Flotillin Adi Liv Tubulin GFP Tubulin GFP Tubulin

47 Improvement of glucose uptake and insulin tolerance in Ade-cav-1 injected JYD mice A Blood glucose (mg/dl) * * * Ade-GFP Ade-cav-1 C p/sec/cm 2 /sr x B % of initial blood glucose * Time (min) * Ade-GFP Ade-cav D Ade-GFP Ade-cav-1 80 Time (min)

48 Caveolin-1 status of the skeletal muscle might influence insulin sensitivity in the aged organism and might contribute to prevent or delay the appearance of type 2 diabetes

49 Exercise and Caveolin-1 Status?

50 Can in vivo expression of caveolin-1 be increased by exercise training?

51 Exercised C57BL/6 Y (6-8 wks), O (68-75 wks), O-ex (68-75 wks) Mus Liv Y O OE Y O OE IR GLUT-4 Cav-1 Cav-3 tubulin

52 Expression of caveolins and insulin related proteins in EDL muscle during resistance exercise B A CON RE AE IRβ GLUT-4 Percent of CON (%) * * * CON RE AE * IRβ GLUT-4 Cav-1 Cav-3 CAV-1 CAV-3 C CON RE AE TUBULIN SOL EDL

53 Effect of exercise on caveolin-1 status in Human study Exercise type : 100m and 1500 m swimming Participants : 14 male penthlon athletes Samplings : Deltoid muscle and Vastus lateralis muscle biopsy before and after the exercise Purpose : modulation of glucose utilization

54 Kim et al., Eur J Appl Physiol (2009) 106:

55 Caveolin-1 and Hormone Sensitivity? 1. Sex hormone effects

56 Glucose and insulin tolerance test of male and female JYD mice Body weight (g) male female 10 0 JYD Male JYD Female Age (weeks) C Blood glucose (mg/dl) * * * JYD male JYD female * % of initial blood glucose * ** * JYD male JYD female * * Time (min) Time (min)

57 Expression level of insulin signaling molecules and caveolin in male and female JYD mice A IRβ ADI MUS LIV PAN M F M F M F M F B 1.2 IRβ C 1.2 AKT/PKB AKT/PKB 1 1 GLUT-4 Cav-1 Cav-3 Tubulin Fold change ADI MUS LIV PAN Fold change ADI MUS LIV PAN Fold change D E F GLUT-4 Fold change Cav-1 Fold change Cav-3 ADI MUS LIV PAN ADI MUS LIV PAN 0 ADI MUS LIV PAN

58 FDG-PET of whole body glucose uptake M MG F %ID/g 4 0 Oh et al (2011) Modulation of Insulin Sensitivity and Caveolin-1 Expression by Orchidectomy. Molecular Medicine. 17(1-2):4-11.

59 Caveolin-1 and Hormone Sensitivity? 2. Growth Hormone Effects

60 Aging dependent growth hormone sensitivity Growth hormone & Insulin sensitivity TYPE 2 DM Sarcopenia P GHR /IGF-IR P JAK IRS IR P TYPE 2 DM STAT IRS P P SHC STAT N Transcription of target genes PI3K AKT GSK GLUT translocation MAPK Proliferation / Differentiation

61 Growth hormone responses in skeletal muscle % of CON CON GH treated IR G4 C1 C3 * * P-STAT GHR bcl tubulin 200 Cav-1 0 CON Ex G.H Ex+G.H

62 Caveolin-1 and Hormone Sensitivity? 3. Glucocorticoid hormone effects

63 Dexamethasone inhibits caveolin-1 in C2C12 myotubes A B C

64 Caveolin-1 status in muscle tissues is sensitive to hormonal status and might be responsible for muscle differentiation and age dependent muscle loss

65 Summary Q1. A1. Can Regulation caveolins of insulin regulate response the insulin signaling in in skeletal skeletal muscle muscle cells as by in caveolin adipocytes? status Q2. A2. Why Development we can not and develop Characterization lean type non of obese ageing dependent type 2 diabetic JYD animal DM model? Q3. A3. Can Potential caveolins role for regulate skeletal the muscle insulin caveolin-1 resistance in ageing dependent type 2 diabetic in JYD JYD model? Q4. A4. Is Exercise in vivo expression type and muscle of caveolin fiber specific increased induction of caveolin-1 expression for by insulin exercise sensitivity training? Q5. A5. Can Modulation sex hormone of Insulin modulate Sensitivity insulin and sensitivitiy Caveolin-1 of Expression by Gonadectomy in Non-Obese Type 2 JYD Diabetes mice?

66 Further Questions Caveolin-1 in Stem Cell Differentiation Cancer Development Longevity

67 Inhibitory effect of caveolin-1 on adipogenic differentiation in young hmsc Ad /GFP Ad / Cav-1 MOI adipogenesis Adipogenesis (%) Park et al (2005) Mechanism of Aging and Development 0 MOI Mock Cav-1

68 Caveolin-1 as an index of wound healing Rhim et al (2010). Molecular Medicine 16:

69 Modulation of wound healing by caveolin-1 status in human corneal epithelium after excimer laser photoablation surgery Ji-Heon Rhim 1, Jae Hoon Kim 2, Jae Chan Kim 2* and Sang Chul Park 1

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72 Summary of Variations detected in caveolin -1 Exon Locus Amino acid change Normal(=62) Frequency Centenarian(=65) intron exon exon % 0.00% - Val->Ile 4.84% 0.00% 1.61% 0.00%

73 Caveolin and aging Cellular aging mechanism 1. Mitogenic response 2. Intracellular trafficking 3. Polarity 4. Metabolic response by hormone sensitivity 5. Microbial infection 6. Structural determination Age related diseases 1. Stem cell differentiation 2. Cancer cell differentiation 3. Wound healing 4. Diabetes 5. Dementia 6. Vascular tone 7. Infectious diseases 8. Muscle aging

74 Collaborators Sung Chun Cho (Gachon Univ) Sung Jin Ryu (NIH) Yoon Sin Oh (Gachon Univ) Jeong Soo Park (Dangook Univ) Kyung A Cho (Chonnam Univ) Young Hoon Son (SNU) Hee Sook Jun (Gachon Univ) Ji Won Yoon (Chicago Univ *) Chang Geun Kim (SSU)