Perturbation of Signal TransductionPathways for Growth Factors and Growth Inhibitors in

Malignant Transformation

3

2

1

-

-

12

3

Different isoforms of PDGF form three types of receptor dimers

PDGF-AB PDGF-BB PDGF-DDPDGF-AAPDGF-CC

Ligandisoform

Extra-cellular

TM

Intra-cellular

Receptortype

In vivo function of PDGF

Embryonal development• kidneys (mesangial cells)• blood vessels (smooth muscle cells, pericytes)• lungs (alveolar smooth muscle cells)• CNS (oligodendrocytes)

Stimulation of wound healing

Regulation of interstitial fluid pressure

Ligand-induced activation of PDGFR causesa conformation switch in the receptor C-terminus

0 10 20 30 40 50 60 700

2500

5000

7500

10000

Wt

ct29

ct46

ct75

Time (min)

Kin

as

e a

ctv

ity

0 10 20 30 40 50 60 700

2500

5000

7500

10000

PDGFR autoinhibition by a C-terminal motif

No PDGF

+ PDGF

Time (min)

Kin

as

e a

ctv

ity

PDGFR

ct75-

ct46-

ct29-

- Y

Control

PDGF

Wt ct46 ct46-D850N

0

10

20

30

40

50

60

70

80

vect

orW

T

D850N

ct46

ct46

-D85

0N

M-R

as Q

71K

Nu

mb

er o

f F

oci

PDGFR activation by mutating the activation loop and the C-terminus

Binding of SH2 domain molecules to autophosphorylatedtyrosines in the PDGF -receptor

P

P

P

P

PP

P

P

P

P

PP

P

PP

P

P

P

P

P

P

P

PP

PLC- mitogenicity

562579581589

716740751763771775778

857

966 97010091021

SHP-2 Grb2/Sos1 Ras Erk MAPK mitogenicity

STAT

RasGAP

Nck

Src mitogenicity

Shc Grb2/Sos1 Ras Erk MAPK mitogenicity

PI3’-kinase Rho, Rac,JNK MAPK actin reorganization and chemotaxis

Grb2/Sos1 Ras Erk MAPK mitogenicity

SS S

S

Akt survival

200

116

97

66

50

37

25

1 2 3

PDGFRPDGFR

RasGAP / FAK

Hsp84

Hsp A5SHP2Stam1

Erk1

Grb2

Alix

Identification of tyrosine phosphorylated proteins after PDGF stimulation

1. Serum-starved cells

2. PDGFR expressing cellsstimulated with PDGF-BB

3. PDGFR expressing cellsstimulated with PDGF-BB

Alix overexpression decreases the rate of PDGFR removal from the cell surface

PAE/PDGFR

PAE/PDGFR-Alix

0

50

100

0 10 30 60PDGF-BB stimulation (min)

Cel

l su

rfa

ce P

DG

FR

(% o

f in

itia

l)

1.0 8.2

Alix

PAE/PDG

FR-

Alix

-actin

PAE/PG

FR

K Monoubiquitylation

Ub

KKKMultimono-ubiquitylation

UbUbUb

Ubiquitylation of proteins

KPolyubiquitylation(via K48 and K63)

UbUb

UbUb

UbUb

UbUb

K

0

2

4

6

0 60 120Time (min)

Ub

iqu

itin

atio

n (

a.u

.)

PAE/PDGFR

PAE/PDGFR-Alix

PDGF-BB (min) 0 30 60 120 0 30 60 120

Ip: PDGFRIb: HA-Ub

Ip: PDGFRIb: PDGFR

Ub-PDGFR

PDGFR

PAE/PDG

FR

PAE/PDG

FR-

Alix

Alix overexpression reduces PDGFR ubiquitination

AlixAlix AlixAlixP

P

Cbl

Ub

Alix may stabilize PDGFR by inducing Cbl degradation

Downregulation of receptor tyrosine kinases

TC-PTP ko wt

PDGF-DD - + - +

IB: P-Tyr

IB: PDGFR

Ligand-activated PDGF -receptor is hyperphosphorylated in TC-PTP knockout cells

0

2

4

6

8

10 wt

TC-PTP ko

rela

tive

rec

epto

r p

hos

ph

oryl

atio

n

Decreased rate of PDGF -receptor degradation in TC-PTP ko MEFs

Direct detection of recycled PDGF -receptors in TC-PTP ko MEFs

The PDGF -receptor co-localizes with Rab4a after 10 min stimulation in TC-PTP ko MEFs

PDGF -receptor Rab4a-EGFP

No colocalization between Rab11EGFP and PDGFR inTC-PTP ko MEFs after 20 min of stimulation

degradation

TC-PTP?

PDGFRPDGFR

TC-PTPP

XP

ESCRT?P

YP

P

XP

P

P

P

PTP?

Trafficking of PDGF receptors

cc

c cc c

N-glycosylation

O-glycosylation

Glycosaminoglycan chain

c Cysteine residues

P

P

P Phosphorylationsite

Structure of the hyaluronan receptor CD44

Link domainHyaluronan-binding domain

Basic motif

Variant exon (v1-v10)Extracellular

Transmembrane

IntracellularERM

Actin

Co-immunoprecipitation of PDGF ß-receptor and CD44

IP:

IB: PDGFRCD-44 PY99

PDGF-BB: - - + - - + - - +

PDGF-BB: - - + - - + - - +

IP:

rabbit IgG

rabbit IgG

rabbit IgG

PDGFR PDGFRPDGFR

ratIgG

CD-44 CD-44CD-44 ratIgG

ratIgG

IB: CD44 PDGFRPY99

PDGFR

CD44

PDGFR

CD44

Hyaluronan - + +Monoclonal Hermes1, that block hyaluronan binding to CD44, restores PDGF ß-receptor activation

Hermes 1 - - +

PDGF-BB + + +

PY99

PDGFRß

Co-exposure of skin fibroblasts to PDGF-BB and hyaluronan inhibits PDGF ß-receptor activation

Hyaluronan (µg/ml) 0 10 25 50 100 0 10 25 50 100PDGF-BB - - - - - + + + + +

PY99

PDGFRß

PTPases account for the hyaluronan-induced PDGF ß-receptor dephosphorylation

Quantification

PervanadateHyaluronan

PDGFR

PDGF-BB

PY99

1 1 10 20 100 220 55 225

Cross-talk between CD44 and the PDGF ß-receptor

CD44-receptor

CD44

-receptor

PPP

PPTPinactive

PP

PP

PDGF-BBPDGF-BB

PTPactive

Signaling

Hyaluronan

Modulated signaling

PDGF in disease

Fibrotic conditions• lung fibrosis• glomerulonephritis• liver cirrhosis• myelofibrosis

Atherosclerosis

Malignancies• autocrine stimulation (glioblastoma, sarcoma)• paracrine stimulation (cancers)

Dermatofibro-sarcomaprotuberans

Chronic mono-myclocytic leukemia

Hyperoesinophilicsyndrome

Gastrointestinalstromal tumor Glioblastoma

PDGF antagonists

PDGF aptamer

-DNA molecule whichbinds PDGF B-chainwith high affinity

-specific

-expensive, cumbersometo administer

STI571 (Glivec, imatinib)

-LMW inhibitor of PDGFreceptors, c-Kit, Abl,and Arg

-not absolutely specific

-inexpensive, easy toadminister

Difference in STI571(Glivec/imatinib) sensitivitybetween different glioblastoma cultures

Clustering of mRNA profiles of glioblastoma cell cultures

PDGF1

32

Effects of PDGF in tumors

bloodvessel

stromal fibroblast

lymphvessel

tumorcells

Effect of anti-PDGFR and anti-VEGFR treatment on B16 melanoma tumor growth

PDGF and the interstitial fluid pressure (IFP) of tumors

•PDGF -receptor activation increases IFP in normal connective tissue

•Many solid tumors display increased IFP

•Increased tumor IFP prevents drug uptake into tumors by decreasing transcapillary transport

•Many solid tumors express PDGF receptors on pericytes and stroma cells

Src

PLC-

Shc

PI3'- kinase

SHP-2

Grb2/Sos1

mitogenicity

mitogenicityRas MAP kinaseGrb2/Sos1

actin reorganization /chemotaxis/migration

Rho, RacAkt

mitogenicityRas MAP kinaseGrb2/Sos1

mitogenicityRas MAP kinase

/ mitogenicityactin reorganization

survival

Binding sites for signal transduction molecules

on the PDGFR-

0.5

1

1.5

-1

-2

-3

-4

-0.5

-1.5

-2.5

-3.5

010 20 30 40 50 60 70

Regulation of Interstitial fluid homeostasis

by the PDGFR- via PI3'kinase

mice with wild type PDGFR-

subdermal injectionof PDGF-BB

injection of C48/80

interstitial fluid pressurepif [mmHg]

time[minutes]

mice with PI3´K binding site mutant PDGFR-

Forces that regulate transcapillarytransport in tissues

b Tumour tissuea Normal tissue

Blood vessel

Net outwardfiltration pressure1-3 mm Hg

Net inward oroutward pressure(-18-2 mm Hg)

COPIF

8 mm HgPIF

-1 to -3 mm HgCOPIF

20 mm HgPIF

10 -30 mm Hg

b Tumour tissuea Normal tissue

b Tumour tissue

Structural differences between normal and tumourtissues that affect interstitial fluid pressure

PDGF and tumor IFP

Tumor-derived PDGF contributes to increased tumor IFP•

- decreases tumor IFP

- synergizes with chemotherapy of tumors

Treatment with PDGF antagonist•

- increases drug uptake in tumors

AcknowledgementsLICR, Uppsala Uppsala University

Bengt WestermarkLena Claesson-WelshKristofer Rubin

Linköping UnivÅke WastesonKarolinska InstituteMonica NisterArne ÖstmanChrister BetsholtzLund Univ. in MalmöLars RönnstrandGothenburg Univ.Keiko FunaLICR, StockholmUlf Eriksson

Johan Lennartsson Piotr Wardega Lotti Rorsman Alexandra JurekCarina Hellberg Aga Koslowska-Wardega Susann Karlsson Christian SchmeesEvi Heldin Lingli LiRainer Heuchel Monica Krampert Shioto Suzuki Aive Åhgren

Ken AmagasakiGudrun BäckströmFederica ChiaraJean-Baptiste DemoulinSimon EkmanMasao FuruhashiYuko HasumiDaniel HägerstrandAnders KallinKaska KowanetzRonggui LiCamilla LoomanKristian PietrasAkira ShimizuTobias Sjöblom