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Wallace McKeehan Center for Cancer & Stem Cell Biology
Forty Years of FGF: Regulator of Cellular &Metabolic Homeostasis
F
IIII
III IIIH
SP
G
F
PLC-
Grb2
SHP2PKC
?
sosRas
Raf
MEK
ERK1/2
Grb2
SN
T1 P
I3K
AK
T
PLC-
sos SNT1
The Klothos
Gordon Sato, The Mangrove Man
Gospodarowicz D, Jones KL, Sato G. 1974Purification of a growth factor for ovarian cells from bovine pituitary glands. PNAS 71:2295
Gospodarowicz D. 1975Purification 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
FGF22 FGF19 FGF21 FGF23
FGF11 FGF12
FGF16 FGF17 FGF18
FGF3 FGF5FGF1 FGF2 FGF4
FGF20
FGF6 FGF7 FGF8 FGF9 FGF10
Continued Discovery ThroughHomology & Genome Sequencing:
22 FGF Homologues
18 High Affinity FGFR Activators
Four TransmembraneTyrosine Kinase ReceptorsNumerous Splice Variants:
FGFR1-4
Blue: Positive ChargeRed: Negative Charge
White: Neutral
Structure, Mechanismof Assembly & Signaling
of the FGFR Complex
II
II
III
II
III
F
III III
FF
Pantoliano…Sisk Biochem. 1994
Spivak-Kroizman…Schlessinger 1994
DiGabriele…Hendrickson, Nature 1998
FII
III
II
III
F
Plotnikov…Mohammadi, Cell 1999
Kan…McKeehan, JBC 1996
McKeehan, Wang, Kan, PNAR 1998
Xu…McKeehan, JBC 1992Kan…McKeehan, Science 1993
F
II
III
II
III
F
III
F
Yayon…Ornitz, Cell 1991
II
III
II
III
F
F
F F
Venkataraman…Sasisekharan, PNAS 1996Moy…Powers, Biochem. 1997
+++++
IIII
F
IIIIII
F
II
IIF
III
II
+
Schlessinger….Mohammadi, Cell 2000
Pelligrini…..Blundell, Nature 2000
IIIIF
IIIIII
F
Kan…McKeehan, JBC 1996McKeehan, Wang, Kan, PNAR 1998
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
Conformational transmembrane communication between ecto and intracellular domains
How Can FGF-dependent Change Outside Be Transmitted Across the Membrane?
Next Generation: Are Intracellular Substrates/AdaptorsOrganized and Waiting for Conformational Derepression?
FIIII
III III
HS
PG
F
PLC-
Grb2
SHP2PKC
?
sos Ras
Raf
MEK
ERK1/2
Grb2
SN
T1 P
I3K
AK
T
PLC-
sosSNT1
F F
Role of Heparan Sulfate
nIdoA/GlcA GlcN
Coreprotein
OCOO-
OX
OH OO
CH2OX
NY
OX OCOO- Linker
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
2-O-, 2-N-, 3-O- and 6-O-sulfates,2-N-acetyl and epimerization
FGF
HS
HS proteoglycan
FGFR
Is There Structural Specificity in Heparan SulfateBeyond Variations in Charge Density?
Purification of an undersulfated 7,8-S-octasaccharide mixture
(7,8-S-OctaF7) by affinity chromatography with FGF7
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.
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).
αKl1 αKl2N C
βKl1 βKl2N C
αKlotho (1014aa)βKlotho (1043aa)
The Klotho Co-factors: single pass transmembraneproteins involved in endocrine FGF activities
SS TM
Similarities to Heparan Sulfate (HS):Independent binding to eFGFs or FGFR
Direct participation in the FGFR complex with both FGF and FGFRDeterminants 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 ofHS or Klothos play a role in signaling?
Development and Adult Homeostasis
Modes of Function:Autocrine, Paracrine, Endocrine
Development: Autocrine and ParacrineControl: Changing, Short-lived Cell Autonomy,
Transcription rate-limiting
Adult Tissue Homeostasis: Largely ParacrineControl: Partition of FGF and FGFR between Cells,
Activity, not transcription rate-limitingAutocrine is Pathological
Intracrine (FGF, FGFR or FGF-FGFR)?
Is there tissue and target cell specificity among the 18 FGFsand 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?
FGF 7FGF10
FGR2b
FGFR3
FGF9
Epithelium
Stroma
Heparan sulfate
Subversion and Autocrine Switch of Canonical Matrix-controlled Short Range Paracrine FGF Signaling Results in Pathologies
Loss of R2bEctopicFGFR1
AutocrineSwitch
Cancer
Feng, Wang, Matsubara, Kan, McKeehan.Fibroblast growth factor receptor 2 limitsand receptor 1 accelerates tumorigenicityof prostate epithelial cells. Cancer Res. 1997
FGFRSHP2SHIP2ERK2IRS4FRS2PLCλ
RSK2FynFAKShcAP85αP85β
Paxillin
PTPN18CDK2 (Tyr15)
EmerinZRF1LAP2
SAP102
growth/tumor suppression, nuclear-cytosol interplay,
cell structural maintenance
FGFR2IIIb FGFR1IIIc
growth promotion,cell survival, adhesion,motility
Overlapping and Distinct TyrP Targets Between:
Luo et al. Novel phosphotyrosine targets of FGFR2IIIb signaling. Cell Signal 2009
Hepatocyte FGFR4
Hepatocyte Cyp7a
Cholesterol to Bile Acids
2000: First implication of FGF signaling in regulating metabolic circuits: Cholesterol/bile acid homeostasis
Time after partial hepatectomy (hr)0 24 48 72 96 120144168
1
2
3
4
5
0102030405060708090
0 24 48 72 96120144168
+/+-/-
+/+-/-
A B
Yu, Wang, Kan…McKeehan. Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. JBC 2000
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 regulatorKharitonenkov et al., JCI 2005
2005:Klothos impact metabolism & FGF signaling
The FGF15/19-FGFR4 Axis:An enterohepatic regulatorof cholesterol/bile acid homeostasis
Inagaki et al., Cell Metab. 2005
Evolution of the Endocrine FGFS
D.D. Moore, Science 2007
Steroids
Steroidreceptors
Paracrine FGFsFGF7,9,10, others
heparan sulfates
FGFR2IIIbFGFR3
Cellularcompartmentalhomeostasis
FGF21 effectorshave expanded!
Hepatocyte FGFR4 Has Multiple EffectsBeyond 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.
Hepatocytes Hepatoma cells
R4 R4 R1
Resident hepatocyte FGFR4 limits hepatocarcinogenesis while ectopic hepatocyte FGFR1 accelerates it
Huang, et al. Ectopic activity of fibroblast growth factor receptor 1 in hepatocytesaccelerates hepatocarcinogenesis by driving proliferation and vascular endothelialgrowth factor-induced angiogenesis. Cancer Res 2006
Huang, et al. Resident hepatocyte fibroblast growth factor receptor 4 limitshepatocarcinogenesis. Mol Carcinog. 2008
WT R1TG WT R1TG
Ectopic FGFR1 in Hepatocytes
FGFR4 Knockout
Normal
liver
0
0.5
1
1.5
2
2.5
Rel
ativ
e E
xpre
ssio
n
of
KL
B
Hepatoma
FGFR4-/-
Hepatoma
P=0.0018
0
20
10
40
Ap
op
tosi
s(A
550
x 10
0)
30
KLB construct 0 0.04 0.2 1 5
FGFR4-/-/FGFR4ecto
FGFR4-/-
bklotho (KLB) is reduced in human & mouse hepatomas
-8
-6
-4
-2
0
2
4
6
8
Rel
ativ
e E
xpre
ssio
n K
LB
Normal LiverHepatoma
Human SIB-CleanEX DbGSE7307;2109
Restoration of KLB and FGFR4 to KLB- and FGFR4-deficientmouse hepatoma cells induces apoptosis
Without comparablechanges in FGFR4
Rel
ativ
e E
xpre
ssio
nKLB drops in liver after partial hepatectomy and recovers during restoration
Day after partial hepatectomy
0
2
4
6
8
Cel
l N
um
ber
x 1
0-5 293 iR4cKLB
HEK293 KLB Constitutive(c) FGFR4 Induced(i) cKLB + iFGFR4
iR4cKLB
(R4)cKLB
iR4cKLB
F1
iR4cKLBF19
iR4F1
iR4F19
iR1cKLB
iR1cKLB
F1
iR1cKLBF19
KLB partners with FGFR4 to inhibit cell population growth via apoptosisApoptosis 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?
F
IIII
III III
HS
PG
F
PLC-
Grb2
SHP2PKC
?
sos Ras
Raf
MEK
ERK1/2
Grb2
SN
T1 P
I3K
AK
T
PLC-
sosSNT1
Klo
tho
s
cFIIII
III IIIH
SP
GcF
PLC-
Grb2
SHP2PKC
?
sos Ras
Raf
MEK
ERK1/2
Grb2
SN
T1 P
I3K
AK
T
PLC-
sosSNT1
cFF1
eF
Local
Canonical(c) FGF Paracrine/Autocrine
Signaling Endocrine(e) FGF Signaling
Cellular Homeostasis(Development & Adult)
Growth, migration, morphogenesisPromotion of Tumor Phenotype
Metabolic HomeostasisInhibition of growth, pro-cell death
Tumor Suppression
cF
DistalLocal
Local
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 expressionof 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 xenograftand FGF19 transgenic hepatocellular carcinoma models. Oncogene 2008
French et al. Targeting FGFR4 inhibits hepatocellular carcinomain preclinical mouse models. PLoS One 2012
Mellor. Targeted inhibition of the FGF19-FGFR4 pathway in hepatocellularcarcinoma; 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 FGFR4-KLB. 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 geneswithout 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.
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 adipocyteFGFR1-mediated adipohepatic communicationduring hepatic stress. Nutr Metab (Lond). 2012]
FGF21/FGFR1-mediated adipo-hepatic communication
Is adipocyte FGFR1 the sole direct mediator of beneficialeffects 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 antibodyactivating 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
IlealFGF19
FGFR4/KLB:Hepatic Cholesterol to Bile AcidsHepatic Lipogenesis
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 conditionsof metabolic extremes and other stressconditions sensed by the liver.
Effects of FGF19/FGF21 on glucosemetabolism are largely an indirect consequence of direct regulation of lipidmetabolism in both liver and adipocytesin both normal and extreme conditions.
Conclusion
Dr. Chundong YuProfessor BiologyXiamen University
Dr. Xinqiang HuangRegulus Therapeutics
Dr. Fen WangProfessor
Center DirectorIBT Texas A&M
US Public Health Service grantsJohn S. Dunn Research FoundationKomen Breast Cancer Foundation
Amgen & Eli Lilly
Dr. Yongde LuoAssistant ProfessorIBT Texas A&M
Chaofeng YangPostdoctoral
UT Southwestern
How could an extracellular co-factor (HS/klotho) alterdiverse intracellular signaling endpoints & phenotypes?
Transmembrane conformational transmission?
F
IIII
III III
HS
PG
F
PLC-
Grb2
SHP2PKC
?
sosRas
Raf
MEK
ERK1/2
Grb2
SN
T1 P
I3K
AK
T
PLC-
sos SNT1
s-k
loth
o
m-k
loth
o
FGF15/19FGF21FGF23
GRB2 SHP-2
SNT1/FRS2
•Activated pathways remarkably similar
•Few P-Tyr substrate sites like other TKR
•Mostly SNT1/FRS2a multi-P adapter
Control cKLB iFGFR4 cKLB + iFGFR4
βKlotho (KLB) partners with FGFR4 or FGFR1 to inhibit cell population growth
KLB- and FGFR4-dependent apoptotic cell death is induced by either FGF19 or FGF1
4.5 9 10.5 13.4 21.4 14.5 Apoptosis (%)
cKLBiFGFR4
FGF19 (1)
cKLBiFGFR4
FGF19 (10)
cKLBiFGFR4
FGF19 (102)
cKLBiFGFR4
FGF19 (103)
PI
4.6 9 9.5 10.5 16.5 23.8
HEK293cKLB
iFGFR4
Annexin V
FGF1 (103)FGF1 (102)FGF1 (10)FGF1 (1)
Apoptosis (%)
PI
Luo et al. Metabolic regulator betaKlotho interacts with fibroblast growth factorreceptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation 2010 JBC