Wallace McKeehan Center for Cancer & Stem Cell Biology
Forty Years of FGF: Regulator of Cellular &Metabolic Homeostasis
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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
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Pantoliano…Sisk Biochem. 1994
Spivak-Kroizman…Schlessinger 1994
DiGabriele…Hendrickson, Nature 1998
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III
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Plotnikov…Mohammadi, Cell 1999
Kan…McKeehan, JBC 1996
McKeehan, Wang, Kan, PNAR 1998
Xu…McKeehan, JBC 1992Kan…McKeehan, Science 1993
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Yayon…Ornitz, Cell 1991
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Venkataraman…Sasisekharan, PNAS 1996Moy…Powers, Biochem. 1997
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Schlessinger….Mohammadi, Cell 2000
Pelligrini…..Blundell, Nature 2000
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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?
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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
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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
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bklotho (KLB) is reduced in human & mouse hepatomas
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Human SIB-CleanEX DbGSE7307;2109
Restoration of KLB and FGFR4 to KLB- and FGFR4-deficientmouse hepatoma cells induces apoptosis
Without comparablechanges in FGFR4
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nKLB drops in liver after partial hepatectomy and recovers during restoration
Day after partial hepatectomy
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HEK293 KLB Constitutive(c) FGFR4 Induced(i) cKLB + iFGFR4
iR4cKLB
(R4)cKLB
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iR4cKLBF19
iR4F1
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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?
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cFF1
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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 disrupt roles in 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
FGF21Ileal FGF19 is under post-prandial bile acid-control.
FGF21 is significant only under conditions of metabolic extremes as starvation and obesity and a wide range of conditions causing systemic and tissue stress.
Liver has been the major notable source of FGF21 during metabolic stress.
FGF21 binds and activates the FGFR1-KLB complex, but not the FGFR4-KLB complex.
FGFR1 is the major FGFR isotype in adipocytes where KLB is notably expressed.(FGFR1 is not significant in hepatocytes where FGFR4-KLB dominates)
FGF19 binds and activates both FGFR4- and FGFR1-KLB complexes************************************************************************************
Are there non-hepatic sources of FGF21?
Is adipocyte FGFR1-KLB the primary direct target of FGF21 during metabolic stress? Does adipocyte FGFR1 account for the beneficial effects of FGF21 and FGF19 on obesity and diabetes?
What is the role of FGF21 activation of adipocyte FGFR1-KLB in alleviation of stress?
Liver:
Starvation, obesity, chemical and genetic disruptions (LKB1, p53, MST1/2, SAV1 and PTEN), loss of liver mass (partial hepatectomy), hepatitis, fatty liver, cirrhosis, hepatoma. Yang et al. BMC Gastroenterology 2014************************************************************************
Skeletal, heart, gastrocnemius muscle: Mitochondrial respiratory, defects, autophagy/mitophagy defects, chronic exercise, Fe-S cluster defect. Suomalainen et al. Lancet Neurol 2011 ; Yang et al. Clin Endocrinol 2011 ; Cuevas-Ramos et al. PLoS One 2012 ; Kim et al. Nat Med 2013 ; Crooks etal. Hum Mol Genet 2014**********************************************************
Brown fat: Cold stress, fetal-neonatal transition.Hondares et al. Cell Metab 2010 ; J Biol Chem 2011 ***********************************************************
Pancreas: Pancreatis.Johnson et al. Gastroenterology 2009 ; Am J Physiol Endocrinol Metab. 2014 ***********************************************************
Are there non-hepatic sources of FGF21?FGF21 is induced by diverse sources of stress in diverse tissues
Diverse Transcriptional Regulators of FGF21:
PPARα/γ, RAR, ChREBP, SREBP1c, LXR, STAT3/5, p53, ATF4, MIST1
Is adipocyte FGFR1-KLB the primary direct target of FGF21 during metabolic stress?
Poising transcriptional activity of hepatic lipogenic genes without much effect on adipocyte genes.
Indirect increase in hepatic steatosis concurrent with increase in hepatic lipogenic genes without much effect on adipocyte gene expression.
Elevation of triglyceride and NEFA without effect on glucose or ketone bodies under starvation conditions concurrent with an increase in adipocyte lipase activity.
Ablation of FGFR1 in Adipocytes
Under stress FGF21 is induced in liver. FGF21 activates adipose FGFR1 that imposes restrictions on adipocyte lipolysis and indirectly hepatic lipogenesis.
This attenuates extent of potentially damaging hepatic steatosis that often occurs during hepatic stress caused by starvation.
Lipolysis and lipogenesis are normally tightly coupled inversely with each other and to glucose and ketone body metabolism.
Uncoupling may mete out and extend lipid reserves for neural fuels (glucose and ketone bodies) during metabolic extremes and other conditions causing hepatic stress.
This mechanism may underlie the beneficial effects of FGF21 (and in part FGF19) under metabolic extremes as well as other sources of hepatic stress.
FGF21/FGFR1-mediated adipo-hepatic communication
Is adipocyte FGFR1 the direct mediator of the beneficialeffects of FGF21 under stress of obesity and diabetes?
Adipocyte-specific ablation of FGFR1 indicates that adipocytes via FGFR1 account for effects of FGF21 and likely the 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
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
Adipose FGFR1 deficiency also abolishes weight loss, glucose and energy regulation by FGF19 in the obese
"...while subtle differences exist in vitro, the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance." Adams...Kharitonenkov PLos One 2012.
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
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
Recap, Summary Overall
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
