fgfgrc022814 - Texas A&M University

Report
Forty Years of FGF:
Regulator of Cellular &
Metabolic Homeostasis
Wallace McKeehan
Center for Cancer & Stem Cell Biology
F
HSPG
F
PI3K
AKT
sos
The Klothos
Grb2sos Ras
PKC
Raf
MEK
?
ERK1/2
Gordon Sato, The Mangrove Man
Gospodarowicz D, Jones KL, Sato G. 1974
Purification of a growth factor for ovarian cells from bovine pituitary glands. PNAS 71:2295
Gospodarowicz D. 1975
Purification of a fibroblast growth factor from bovine pituitary. JBC 250:2515
Maciag T, Mehlman T, Friesel R, Schreiber AB. 1984.
Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain. Science 31;225:932.
Gospodarowicz D, Cheng J, Lui GM, Baird A, Böhlen P. 1984.
Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography:
identity with pituitary fibroblast growth factor. PNAS 81:6963.
1985-1986: Cloning of FGF1 and FGF2
1989-1993: Cloning and diversity of FGFR tyrosine kinases via splice variants
1993-2002: Structure of FGFR kinases and interaction with heparan sulfate
Structure, Mechanism
of Assembly & Signaling
FGF1
FGF2
FGF3
FGF4
FGF5
of the FGFR Complex
Continued Discovery Through
Homology & Genome Sequencing:
FGF6
FGF7
FGF11 FGF12
FGF8
FGF9
FGF16
FGF17 FGF18
FGF10
22 FGF Homologues
18 High Affinity FGFR Activators
Four Transmembrane
Tyrosine Kinase Receptors
Numerous Splice Variants:
FGFR1-4
FGF20
FGF22
Blue: Positive Charge
Red: Negative Charge
White: Neutral
FGF19
FGF21
FGF23
F
F
Yayon…Ornitz, Cell 1991
Xu…McKeehan, JBC 1992
Kan…McKeehan, Science 1993
Pantoliano…Sisk Biochem. 1994
F
Spivak-Kroizman…Schlessinger 1994
Venkataraman…Sasisekharan, PNAS 1996
Moy…Powers, Biochem. 1997
F
McKeehan, Wang, Kan, PNAR 1998
Kan…McKeehan, JBC 1996
F
DiGabriele…Hendrickson, Nature 1998
Plotnikov…Mohammadi, Cell 1999
Kan…McKeehan, JBC 1996
McKeehan, Wang, Kan, PNAR 1998
Schlessinger….Mohammadi, Cell 2000
Conformational Model of Control and Activation
(Derepression) of the FGFR Complex
Pre-existent unliganded symmetric complex of 2:2 FGFR-HS
Conformational maintenance of dependence on FGF for kinase derepression by
transphorylation by heparan sulfate in the ectodomain
Pelligrini…..Blundell, Nature 2000
Conformational transmembrane communication between ecto and intracellular
domains
How Can FGF-dependent Change Outside
Be Transmitted Across the Membrane?
Next Generation: Are Intracellular Substrates/Adaptors
Organized and Waiting for Conformational Derepression?
F
HSPG
F
PI3K
AKT
sos
Grb2 sos Ras
PKC
Raf
MEK
?
ERK1/2
Role of Heparan Sulfate
COOO
COOOH
CH2OX
O
Linker
O OX
O
OX
IdoA/GlcA
NY
GlcN
FGF
Core
protein
HS proteoglycan
n
2-O-, 2-N-, 3-O- and 6-O-sulfates,
2-N-acetyl and epimerization
HS
FGFR
1. Matrix and Membrane:
Reservoir, Stabilizer, FGF Delivery System
2. Integral Component of the FGFR Complex:
Specificity for FGF
Assembly of the oligomeric complex
Negative restriction of the unliganded dimeric complex
Stabilization of the derepressed, ligand-activated complex
Is There Structural Specificity in Heparan Sulfate
Beyond Variations in Charge Density?
7,8-S-OctaF7, which has anticoagulant activity and likely the motif containing a 3-O sulfate,
specifically supports high affinity FGF7 binding and mitogenesis in cells expressing FGFR2IIIb.
It failed to support high affinity FGF1 binding and mitogenesis.
Purification of an undersulfated
7,8-S-octasaccharide mixture
(7,8-S-OctaF7) by affinity
chromatography with FGF7
Hypothetical deduced structure of 7,8-S-OctaF7. The x, y or z could be a sulfate,
preferably 6-O-sulfate with the other two as hydrogens (8-S), or x, y and z may all be
hydrogens (7-S).
The Klotho Co-factors: single pass transmembrane
proteins involved in endocrine FGF activities
αKlotho
N
(1014aa)
βKlotho
N
(1043aa)
SS
αKl1
αKl2
βKl1
βKl2
C
TM
C
Similarities to Heparan Sulfate (HS):
Independent binding to eFGFs or FGFR
Direct participation in the FGFR complex with both FGF and FGFR
Determinants of specificity for eFGFs
Are HS and Klothos at play in the same FGFR complex?
Do HS or Klotho co-factors alter quality of the FGFR signal?
Does the protein core of HS or the intracellular domain of
HS or Klothos play a role in signaling?
Development and Adult Homeostasis
Modes of Function:
Autocrine, Paracrine, Endocrine
Development: Autocrine and Paracrine
Control: Changing, Short-lived Cell Autonomy,
Transcription rate-limiting
Adult Tissue Homeostasis: Largely Paracrine
Control: Partition of FGF and FGFR between Cells,
Activity, not transcription rate-limiting
Autocrine is Pathological
Intracrine (FGF, FGFR or FGF-FGFR)?
Is there tissue and target cell specificity among the 18 FGFs
and the 4 tyrosine kinase receptors and variants?
Specificity set by:
1. Paracrine partition of FGF and the FGFR complex between cells.
2. Endocrine partition of FGF and FGFR complex between organs.
3. Cell-specific co-receptors HS and klothos.
Do different FGFR isotypes have exhibit different signaling
endpoints in the same context?
Subversion and Autocrine Switch of Canonical Matrix-controlled
Short Range Paracrine FGF Signaling Results in Pathologies
Epithelium
Loss of R2b
Ectopic
FGFR1
Autocrine
Switch
Cancer
FGR2b
FGF 7
FGF10
FGF9
FGFR3
Heparan
sulfate
Stroma
Feng, Wang, Matsubara, Kan, McKeehan.
Fibroblast growth factor receptor 2 limits
and receptor 1 accelerates tumorigenicity
of prostate epithelial cells. Cancer Res. 1997
Overlapping and Distinct TyrP Targets Between:
FGFR2IIIb
PTPN18
CDK2 (Tyr15)
Emerin
ZRF1
LAP2
SAP102
FGFR1IIIc
FGFR
SHP2
SHIP2
ERK2
IRS4
FRS2
PLCλ
growth/tumor suppression,
nuclear-cytosol interplay,
cell structural maintenance
RSK2
Fyn
FAK
ShcA
P85α
P85β
Paxillin
growth promotion,
cell survival, adhesion,
motility
Luo et al. Novel phosphotyrosine targets of FGFR2IIIb signaling. Cell Signal 2009
2000: First implication of FGF signaling in regulating metabolic
circuits: Cholesterol/bile acid homeostasis
A5
4
Hepatocyte FGFR4
+/+
-/-
3
2
1
0 24 48 72 96 120144168
B 9080
+/+
-/-
70
60
50
40
30
20
10
0
0 24 48 72 96 120144168
Time after partial hepatectomy (hr)
Hepatocyte Cyp7a
Cholesterol to Bile Acids
Yu, Wang, Kan…McKeehan. Elevated cholesterol
metabolism and bile acid synthesis in mice lacking
membrane tyrosine kinase receptor FGFR4. JBC 2000
Evolution of the Endocrine FGFS
The FGF15/19-FGFR4 Axis:
An enterohepatic regulator
of cholesterol/bile acid homeostasis
Early 2000:
FGFR4 involved in cholesterol/bile acid
metabolism. Yu et al. JBC 2000
Late 2000:
Mutation in FGF23 involved in mineral
metabolism. ADHR Consortium, Nat
Genetics 2000
2002:
FGF19 impacts metabolic rate and
adiposity. Tomlinson et al., Endocrinol
2002
2005:
FGF21 is a metabolic regulator
Kharitonenkov et al., JCI 2005
Inagaki et al., Cell Metab. 2005
2005:
Klothos impact metabolism & FGF
signaling
Steroids
FGF21 effectors
have expanded!
Steroid
receptors
Paracrine FGFs
FGF7,9,10, others
heparan sulfates
FGFR2IIIb
FGFR3
Cellular
compartmental
homeostasis
D.D. Moore, Science 2007
Hepatocyte FGFR4 Has Multiple Effects
Beyond Hepatic Cholesterol/Bile Acid Metabolism
1. Limits extent of toxic liver injury and fibrosis
2. Modulates hepatic lipid and glucose metabolism
3. Supports fatty liver in obesity or starvation
4. Reported as both hepatoma promoter and hepatoma suppressor
Which effects beyond bile acid metabolism occur directly
within hepatocytes due to FGF19-FGFR4-bklotho signaling
is unclear.
Resident hepatocyte FGFR4 limits hepatocarcinogenesis
while ectopic hepatocyte FGFR1 accelerates it
Hepatoma cells
Hepatocytes
Huang, et al. Ectopic activity of fibroblast growth factor receptor 1 in hepatocytes
accelerates hepatocarcinogenesis by driving proliferation and vascular endothelial
growth factor-induced angiogenesis. Cancer Res 2006
Huang, et al. Resident hepatocyte fibroblast growth factor receptor 4 limits
hepatocarcinogenesis. Mol Carcinog. 2008
FGFR4 Knockout
Ectopic FGFR1 in Hepatocytes
WT
R1TG
WT
R1TG
bklotho (KLB) is reduced in human & mouse hepatomas
Relative
Expression KLB
8
6
Normal Liver
Hepatoma
4
2
Without comparable
changes in FGFR4
0
-2
-4
-6
Human SIB-CleanEX DbGSE7307;2109
2.5
2
1.5
1
0.5
0
P=0.0018
Apoptosis
(A550 x 100)
Relative Expression
of KLB
-8
40
FGFR4-/-/FGFR4ecto
FGFR4-/-
30
20
10
0
KLB
construct
0 0.04 0.2
1
5
Restoration of KLB and FGFR4 to KLB- and FGFR4-deficient
mouse hepatoma cells induces apoptosis
Relative Expression
KLB drops in liver after partial hepatectomy and recovers during restoration
Day after partial hepatectomy
Cell Number x 10-5
HEK293
8
6
293
KLB Constitutive(c) FGFR4 Induced(i)
cKLB
iR4
iR4 iR4
iR4
(R4) iR4
cKLB cKLB F19 cKLB F1
F19
cKLB + iFGFR4
iR4
iR1
iR1
iR1
cKLB cKLB cKLB cKLB
F1
F19
F1
4
2
0
KLB partners with FGFR4 to inhibit cell population growth via apoptosis
Apoptosis induced by the FGFR1/4-KLB pair is enhanced by either FGF1 or FGF19
FGF21 is specific for the FGFR1-KLB partner
KLB can confer growth controlling, anti-tumorigenic pro-apoptotic
activity on both FGFR4 and FGFR1 signaling complexes.
This is in addition to the role of KLB in conferring high affinity of
FGFR4 for endocrine FGF19 and FGFR1 for both FGF19 and FGF21.
Direction of pro-apoptotic signaling is likely through KLB-dependent
abrogation of anti-apoptotic AKT and mTOR pathways
How might this KLB-dependent redirection occur?
KLB-dependent abrogation of anti-apoptotic AKT and mTOR pathways?
Canonical(c) FGF
Paracrine/Autocrine
Signaling
Endocrine(e) FGF Signaling
F
F1
HSPG
Local
PKC
Raf
PI3K
Grb2 sos Ras
AKT
PI3K
AKT
sos
Distal
HSPG
Local
Local
eF
F
Klothos
cF
cF
sos
Grb2 sos Ras
PKC
MEK
?
ERK1/2
Cellular Homeostasis
(Development & Adult)
Growth, migration, morphogenesis
Promotion of Tumor Phenotype
Raf
MEK
?
ERK1/2
Metabolic Homeostasis
Inhibition of growth, pro-cell death
Tumor Suppression
Net KLB-directed anti-growth and anti-tumor effects is consistent with primary
function of eFGFs in control of metabolic homeostasis.
Targeting hepatic FGFR4 will have serious effects on metabolic homeostasis,
particularly bile acid metabolism and may have tumor-promoting effects.
Nicholes et al. A mouse model of hepatocellular carcinoma: ectopic expression
of fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am J Pathol 2002
Desnoyers et al. Targeting FGF19 inhibits tumor growth in colon cancer xenograft
and FGF19 transgenic hepatocellular carcinoma models. Oncogene 2008
French et al. Targeting FGFR4 inhibits hepatocellular carcinoma
in preclinical mouse models. PLoS One 2012
Mellor. Targeted inhibition of the FGF19-FGFR4 pathway in hepatocellular
carcinoma; translational safety considerations. Liver Int. 2014
FGF21
In contrast to bile acid-controlled diurnal ileal FGF19, FGF21 is
significant under conditions of metabolic extremes as starvation and
obesity and other sources of organismic stress.
FGF21 binds and activates the FGFR1-KLB complex, but not FGFR4KLB. FGFR1 is not expressed in hepatocytes, but is the major FGFR in
adipocytes where KLB is also expressed. FGF19 binds and activates
both FGFR4- and FGFR1-KLB complexes.
[Yang et al. Differential specificity of endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in complex with KLB. PLoS
One 2012]
•What is the role of adipocyte FGFR1 during metabolic stress
conditions where FGF21 is significant?
•Is adipocyte FGFR1 an additional target of FGF19 and the major or
sole target of FGF21?
•Does FGFR1 account for the beneficial effects of FGF21 and FGF19 on
obesity and diabetes?
Adipocyte-specific ablation of FGFR1 indicates that the adipocyte via
FGFR1 is the (possibly the sole) FGFR target that accounts for
metabolic effects of FGF21 and the extra-hepatic effects of FGF19
•Yang et al. Control of lipid metabolism by adipocyte FGFR1-mediated adipohepatic
communication during hepatic stress. Nutr Metab (Lond). 2012
•Adams & Yang et al. The breadth of FGF21s metabolic actions are governed by FGFR1
in adipose tissue. Mol. Metab. 2012
•Foltz et al. Treating diabetes and obesity with an FGF21-mimetic antibody activating the
βKlotho/FGFR1c receptor complex. Sci Transl Med. 2012
Under normal conditions, adipocyte FGFR1 deficiency
causes an increase in transcriptional activity of hepatic
lipogenic genes without effect on adipocyte genes.
Under starvation conditions the FGFR1 deficiency
indirectly causes an increase in hepatic steatosis
concurrent with an increase in hepatic lipogenic genes
without much effect on adipocyte gene expression.
Under starvation conditions the adipocyte FGFR1 deficiency causes concurrent
elevation of triglyceride and NEFA without effect on glucose or ketone bodies. This
occurs concurrent with an increase in adipocyte lipase activity.
FGF21/FGFR1-mediated adipo-hepatic communication
Under starvation conditions that cause hepatic stress and
steatosis, adipose FGFR1 concurrently imposes restrictions on
adipocyte lipolysis and indirectly hepatic lipogenesis.
This serves to attenuate extent of compensatory hepatic steatosis
that often occurs during hepatic stress.
Lipolysis and lipogenesis are normally tightly coupled to glucose
and ketone body metabolism. Overall lipolysis and lipogenesis
are tightly coupled and inversely related.
Uncoupling these normally tightly linked domains may mete out
and extend lipid reserves for neural fuels (glucose and ketone
bodies) during metabolic extremes and other conditions causing
hepatic stress.
Adipocyte FGFR1-KLB is a target of FGF21 whose primary origin
is hepatocytes and an additional target of FGF19 in addition to
hepatocyte FGFR4.
We speculate this mechanism may underlie the beneficial effects
of both endocrine FGFs under both metabolic extremes of
starvation and obesity as well as other sources of hepatic stress.
[Yang et al. Control of lipid metabolism by adipocyte
FGFR1-mediated adipohepatic communication
during hepatic stress. Nutr Metab (Lond). 2012]
Is adipocyte FGFR1 the sole direct mediator of beneficial
effects of FGF21 in obesity/diabetes (metabolic stress)?
Adipose FGFR1 deficiency abolishes weight loss, glucose
and energy regulation by FGF21 in the obese
Foltz et al. Treating diabetes and obesity with an FGF21-mimetic antibody
activating the βKlotho/FGFR1c receptor complex. Sci Transl Med. 2012
What about FGF19’s effects in the adipocytes?
Adipose FGFR1 deficiency also abolishes weight loss, glucose
and energy regulation by FGF19 in the obese
Thus FGF19 targets adipocytes via FGFR1 with
essentially identical effects to FGF21
FGFR4/KLB:
Hepatic Cholesterol to Bile Acids
Hepatic Lipogenesis
Ileal
FGF19
Conclusion
During normal feeding ileal FGF19 is
likely the normal coordinator of
adipo-hepatic communication in the
lipid metabolism domain.
FGF21 is a hepatokine reserved to
instruct adipocytes under conditions
of metabolic extremes and other stress
conditions sensed by the liver.
Effects of FGF19/FGF21 on glucose
metabolism are largely an indirect
consequence of direct regulation of lipid
metabolism in both liver and adipocytes
in both normal and extreme conditions.
Dr. Chundong Yu
Professor Biology
Xiamen University
US Public Health Service grants
John S. Dunn Research Foundation
Komen Breast Cancer Foundation
Amgen & Eli Lilly
Dr. Xinqiang Huang
Regulus Therapeutics
Dr. Yongde Luo
Chaofeng Yang
Assistant Professor
Postdoctoral
IBT Texas A&M
UT Southwestern
Dr. Fen Wang
Professor
Center Director
IBT Texas A&M
How could an extracellular co-factor (HS/klotho) alter
diverse intracellular signaling endpoints & phenotypes?
Transmembrane conformational transmission?
SHP-2
GRB2
SNT1/FRS2
PI3K
AKT
sos
•Few P-Tyr substrate sites like other TKR
F
F
•Mostly SNT1/FRS2a multi-P adapter
HSPG
m-klotho
FGF15/19
FGF21
FGF23
s-klotho
•Activated pathways remarkably similar
Grb2sos Ras
PKC
Raf
MEK
?
ERK1/2
βKlotho (KLB) partners with FGFR4 or FGFR1 to inhibit cell population growth
Control
cKLB
iFGFR4
cKLB + iFGFR4
KLB- and FGFR4-dependent apoptotic cell death is induced by either FGF19 or FGF1
cKLB
iFGFR4
9
9.5
cKLB
iFGFR4
FGF19 (10)
cKLB
iFGFR4
FGF19 (102)
cKLB
iFGFR4
FGF19 (103)
PI
HEK293
cKLB
iFGFR4
FGF19 (1)
Apoptosis (%)
4.6
10.5
FGF1 (10)
FGF1 (102)
23.8
FGF1 (103)
PI
FGF1 (1)
16.5
Annexin V
Apoptosis (%) 4.5
9
10.5
13.4
Luo et al. Metabolic regulator betaKlotho interacts with fibroblast growth factor
receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation 2010 JBC
21.4
14.5

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