Systemic Iron Homeostasis

FeFe22+

Karin E Finberg M D Ph DKarin E. Finberg, M.D., Ph.D.Assistant Professor of Pathology

Yale School of MedicineBioIron Introductory Course

May 7, 2017

Disrupted Iron Balance Leads to Profound Clinical Consequences

3; 2

013-

4045

FeFe22+

ASH

Imag

e Ba

nk 2

013

Too much: Too little:

A

Free iron accumulates in tissues causing oxidative damage

Impairs production of essential proteins(including hemoglobin)

AnemiaOrgan failure(liver, heart, endocrine glands)

AnemiaImmune DysfunctionCognitive Impairment

Impaired GrowthImpaired Growth

Systemic Iron Balance Must Be Tightly Regulated

Red Blood Cells

Duodenum

Macrophages

Dietary Iron1-2 mg/day

Tf-Fe3+

Plasma

25 mg/day

E i

No known regulated mechanism for iron

excretion

Excess iron

Liver

Body iron content is primarily regulated by modulating intestinal Livermodulating intestinal

iron absorption

The Hormone Hepcidin is a Key Regulator of Iron BalanceSmall peptide secreted by hepatocytes that is detectable in blood & urine

enterocytesdietary iron

macrophages

RBC

hepcidinplasma plasma

Liver

plasma

Limits absorption of

plasma

Limits recycling ofLiverpdietary iron

Limits recycling of iron stores

The Hormone Hepcidin is a Key Regulator of Iron BalanceSmall peptide secreted by hepatocytes that is detectable in blood & urine

enterocytesdietary iron

macrophages

RBC

hepcidinplasma

ferroportin

plasma

ferroportin

Liver

plasma

Limits absorption of

plasma

Limits recycling of Liverpdietary iron

y giron stores

Essential Role of Hepcidin in Iron Balance Shown by Genetics

Systemic iron overloadHepcidin deficiency

Hepcidin overexpressionSevere iron deficiency anemia

Severe iron deposition in organspresenting in 1st – 3rd decade(j il f f h h t i )Loss-of-function mutations

in hepcidin gene (HAMP)

(juvenile form of hemochromatosis)

Hepcidin is Produced Primary by Hepatocytes

Several Physiological Stimuli Modulate Hepcidin Production By Modulating the Transcription of the Hepcidin Gene

Anemia & Hypoxia

Iron loading InflammatoryStimuli

See Concurrent Session IMon 5/8 13:50-15:00Iron Sensing in the Liver

See Concurrent Session VWed 5/10 14:00-15:30Iron, Infection & Inflammation

promoter

Hepcidin

Other emerging regulators:G th h

hepatocyte• Growth hormones• Steroid hormones• Gluconeogenic signals

See Also Featured Podium Presentations Wed 5/10 15:45-17:15

Regulation of Hepcidin Production by IronRegulation of Hepcidin Production by Iron

Anemia & Hypoxia

Iron loading InflammatoryStimuli

promoter

Hepcidin

hepatocyte

Genetic Defects That Impair the Regulation of Hepcidin by Iron Underlie Inherited Iron Overload Disordersby Iron Underlie Inherited Iron Overload Disorders

Mutated HepcidinMutatedGene(s) Phenotype Hepcidin

Defect Systemic Effect

Hepcidin (HAMP)Hepcidin (HAMP)

Hemojuvelin(HFE2, aka HJV)

Juvenile Hemochromatosis

Absent or impaired

Failure to appropriately limitimpaired

induction by iron stores

appropriately limitabsorption of dietary iron

HFE Adult Onset

Transferrin Receptor 2 (TFR2)

Hemochromatosis

More on Hereditary Hemochromatosis from Dr. Pietrangelo this afternoon

Bone Morphogenetic Protein (BMP) Signaling:Key Pathway Promoting Hepcidin Transcription in Hepatocytes

BMPRI

BMP

II

SMAD4

R-SMADR-SMADSMAD4

PR-SMADPP

R l f ifi BMP T 1 tSMAD4SMAD4 Role for specific BMP Type1 receptor isoforms (ALK2, ALK3) and for the common SMAD4 in hepcidin regulation demonstrated in liver-specific KO mice

Hepcidin promoter

p

hepatocyte

nucleus

Multiple BMP Family Member Ligands Induce Hepcidin in VitroBut BMP6 Plays a Key Role in Hepcidin Signaling in Vivo

BMP6 mRNA expression in liver is induced by dietary iron loading

?

Liver sinusoidal

Bmp6

BMPRGlobal loss of Bmp6

BMP6

I II

e s uso daendothelial cell

SMAD4

R-SMADP

• Severe hepcidin deficiency and systemic iron overload

SMAD4

Bmp6 KO in sinusoidal endothelial cells

• Recapitulates hemochromatosis

Hepcidin promoter

• Recapitulates hemochromatosis phenotype of global Bmp6 KO

hepatocyte

nucleus • Mechanism of BMP6 transcriptional regulation by iron not yet clear

Endothelial Cell Production of BMP2 Also Shown to Regulate Hepcidin Productiong p

?

Liver sinusoidal

Bmp2

BMPR

BMP2

I IIBmp2 KO in sinusoidal endothelial cells

e s uso daendothelial cell

SMAD4

R-SMADP

• Hepcidin deficiency and systemic iron overload

SMAD4

• BMP2 expression upregulated in livers of thalassemic mice

Hepcidin promoter

• Further understanding of role BMP2 in systemic iron regulation needed

hepatocyte

nucleus

The Juvenile Hemochromatosis Protein Hemojuvelin (HJV)Augments BMP Signaling for Hepcidin Transcription

HJV is a GPI-anchored protein that acts as a co-receptor for BMPs

BMPRBMP

HJV

Hjv-/-I II

SMAD4

R-SMADP HJV mutations• Severe hepcidin deficiency & Fe overload

JHNeogenin

SMAD4

Hepcidin promoter

HJV may act as a bridge between BMPs and neogenin, a TM receptor that appears to modulate BMP signaling

hepatocyte

nucleus

to modulate BMP signaling

The Hemochromatosis Proteins HFE & Transferrin Receptor 2 (TFR2)Promote Hepcidin Synthesis in the Liver

HFE

Iron-bound (holo-) transferrin

Tf

Fe2+ Tf

TfTf Iron-sensing

• MHC Class I-like membrane protein

• Competes with iron bound-Tf for binding to transferrin receptor 1 (TFR1)

TFR1 HFE TFR2

TfTf Iron sensing

model

• Induces hepcidin when released from TFR1

• Interacts with TFR2 to promote hepcidin??

Hepcidin promoter

Precise mechanism by which HFE and TFR2 stimulate hepcidin production under investigation

hepatocyte

nucleus

under investigation

HFE Appears to Promote Hepatic BMP Signaling

Show blunted BMP signalingHfe-/-

BMP6 BMP6?BMP6

HJV Show blunted BMP signaling response to BMP6 ligand

HFE TFR2

Tf

TFR1

Tf

SMAD4

R-SMADPHfe overexpression in liver

P

SMAD4Increased expression of

BMP target genes

Hepcidin & other BMP target genesThe hemochromatosis proteins may form a membrane-associated complex

hepatocyte

nucleuswith the BMP receptor to promote hepcidin expression

BMP Signaling for Hepcidin Production is Down-regulated by TMPRSS6 (aka Matriptase-2)

TMPRSS6

• Transmembrane serine protease expressed in the liver

by TMPRSS6 (aka Matriptase 2)

BMPR

BMP6HJV TMPRSS6 expressed in the liverHJV

• Cleaves HJV from plasma membrane

SMAD4

R-SMADPP• Inappropriately

elevated hepcidin• Systemic iron

Biallelic TMPRSS6SMAD4

• Regulation of TMPRSS6

ydeficiencymutations

Hepcidin promoter

activity under investigation (Fe status, hypoxia)

hepatocyte

nucleus More on Iron Deficiency Anemia from Dr. Camaschella this afternoon

Regulation of Hepcidin Production by Inflammation

Autoimmune diseasesInfections

Regulation of Hepcidin Production by Inflammation

Certain malignancies

InflammatoryStimuli

Iron retention in macrophages

promoter

Hepcidin

p gand decreased dietary iron absorption contribute toanemia of inflammation(anemia of chronic disease)

hepatocyte( )

More on Anemia of Inflammation from Dr. Ganz this afternoon

Interleukin-6 Plays a Key Role in Inducingth H idi R t I fl ti

LPSIL-1

TNF α the Hepcidin Response to InflammationTNF-α

IL-6

JAK

STAT3STAT3PP STAT3PP

Hepcidin promoter

hepatocyte

nucleus

Hepcidin Signaling in Inflammation Involves Cooperative Activity of the BMP Pathway

LPSIL-1

TNF α p y yTNF-α

Fail to induce hepcidin

IL-6

IL-6

JAK

BMP6 Smad4 liver -/-hepcidin

IL-6• Inflammation can also induce hepcidin

expression independently of the BMP6I II

STAT3STAT3PP

SMAD4

R-SMADP Wild typePre-tx with BMP Type 1

receptor inhibitor

Fail to induce hepcidin

expression independently of the BMP6 ligand

• This may involve other ligands of the t f i th f t β/BMPSMAD4 receptor inhibitor

• A certain level of basal BMP signaling is required for the hepcidin response to IL-6 ( ti l b t SMAD d STAT3

transforming growth factor β/BMP superfamily such as activin B

Hepcidin

(cooperatively between SMAD and STAT3 at hepcidin promoter)

BMP-RE STAT3-RE

hepatocyte

ppromoter

nucleus

BMP RE STAT3 RE

Regulation of Hepcidin Production by ErythropoiesisRegulation of Hepcidin Production by Erythropoiesis

Anemia & Hypoxia

Iron loading InflammatoryStimuli

promoter

Hepcidin

hepatocyte

Hepcidin is Suppressed By Increased Erythropoietic Activity

• Large Volume Blood Loss in Otherwise Healthy Individuals• Disorders of Ineffective Erythropoiesis (e.g. β-thalassemia)

Anemia & Hypoxia

promoter

HepcidinUpregulated absorption of

dietary iron to support increased erythropoiesis

hepatocyte

More on Iron-Loading Anemias from Dr. Viprakasit this afternoon

Elevations in Erythropoietin Correlate with Hepcidin Suppression

EPO

Kid

Hypoxia

EPO

Kidney Bone Marrow RBC

Marked reduction in urinary hepcidin levels

Healthy

Rapid marked increase in ser m EPORapid, marked increase in serum EPOReduction in serum hepcidinAcute hypoxic

exposure(high altitude)

The Effect of Epo on Hepcidin Suppression is Indirectand Requires Erythropoietic Activity By the Bone Marrow

EPO

Kid

Hypoxia XEPO

Kidney Bone MarrowXMarked acute reduction in hepcidin

EPO

Pretreated withmarrow-suppressing

cytotoxic agent

Hepcidin-suppressing effects of Epo abolished

y g

Unclear if hypoxia also exerts a local effect in hepatocytes to regulate hepcidin

Products Secreted by Erythroid Precursors Suppress Hepcidin(“Erythoid Regulators” of Hepcidin Production)

EPO

Kid

Hypoxia

Several secreted

Kidney Bone Marrow

ERFE factors described

Erythroferrone (ERFE)

ERFE

y ( )• C1q-TNF protein family member• mRNA rapidly induced in mouse

bone marrow & spleen during

promoter

Hepcidin

stress erythropoiesis

Hepatocyte

promoter

ERFE Plays an Important Role in Ensuring Iron SupplyDuring Stress Erythropoiesis in vivo

EPO

Fail to acutely suppress hepcidin

Large Volume

Erfe-/- mice

gPhlebotomy

Fail to acutely suppress hepcidinand show a delayed recovery in Hgb levelsand show a delayed recovery in Hgb levels

Erfe-/- mice

Raises hepcidin and decreases iron loading (but does not abolish iron loading)

Ablation of Erfe in thalassemic mice May suggest other erythroid regulators of hepcidinthalassemic mice May suggest other erythroid regulators of hepcidin

Disruption of Proteins Involved in Cellular Iron HomeostasisCan Also Impact Systemic Iron Balance

• Defects within enterocytes can disrupt systemic iron balance despite intact hepcidin regulation

Ferritin H subunit Show increased absorption of dietary ironFerritin H subunit intestinal KO

Show increased absorption of dietary iron and systemic iron loading

(despite hepcidin induction)

Hif2α intestinal KO Show impaired expression of key genes involved in apical and basolateral iron transport in enterocytesresulting in lower levels of iron in plasma and liver

(despite hepcidin downregulation)

Hepcidin-Independent Mechanisms of Ferroportin Regulation Can Impact Systemic Iron BalanceRegulation Can Impact Systemic Iron Balance

• Activation of toll-like receptors (TLRs) on reticuloendothelial macrophages canon reticuloendothelial macrophages can downregulate ferroportin expression and promote hypoferremia

• May have relevance to theMay have relevance to the hypoferremic response to specific pathogens that activate TLRs

Some Closing Thoughts…

• Although we have learned much about molecular mechanisms regulating iron balance, many key questions remain, such as:g g y y q

– How do the hemochromatosis proteins intersect with the BMP pathway to promote hepcidin expression?

– How is BMP6 expression regulated by iron?

– How does ERFE act on hepatocytes to downregulate hepcidin p y g pproduction?

– How do other hormonal regulators impact systemic iron homeostasis?

– How do non-hepcidin dependent mechanisms contribute?

Some suggested reading…

Clement Finch (1915-2010)Chief of HematologyChief of HematologyUniversity of Washington

Acknowledgments

The Many Scientists and CliniciansWho Have Contributed to Advancing Our

Understanding of Systemic Iron Regulation