Androgens and erythropoiesis
Julie McManus
University of Melbourne
Department of Medicine
Austin Health
Androgens
• Naturally
N t ll occurring
i androgens:
d
Testosterone
5α-dihydrotestosterone (DHT)
androsterone
(also non-androgenic steroids 5β- dihydrotestosterone and
etiocholanolone)
• Synthetic analogs:
19-nandrolone
Oxymetholone
Stanozolol
Increased androgens →
increased RBC parameters
19-nandrolone
+EPO
EPO
+19-ND
norethandrolone
Decreased androgens →
decreased RBC parameters
LHRH agonist
no change in serum EPO
Evidence from experimental models of
androgen mediated erythropoiesis (1)
Animal Models:
most studies are old –
↑ CFU-E in BM and spleen cultures
↑ uptake of 59Fe
↑ RBC/ ↑ Hb
• 19-ND → ↑RBC ↑WBC ↑Ter119 cells in
spleen in SAMP mice (Saitoh 1999)
Evidence from experimental models of
androgen mediated erythropoiesis (2)
Cell models:
↑ CFU-E
CFU E
• T, DHT, 5β-DHT (Beckman 1981, Besa 1981) –
but findings inconsistent
• oxymetholone
y
((Kim 2005))
• 19-ND (Saitoh 1999)
↑ proliferation
lif
i erythroblast
h bl
(♀)
• 5α
5α-DHT
DHT (Leberbauer 2005)
Mechanism: via the androgen
receptor
Studies in testicular feminised mouse
- inconsistent
i
i t t findings
fi di
(B k
(Beckman
1981
1981, B
Besa
1981, Byron 1977)
Mechanism : direct action on
erythroid
th id precursors
19-nandrolone
Mechanism: indirect action via
erythropoietic growth factors
• EPO
- results equivocal in both clinical and animal models
- no increase in se EPO in testosterone treated men
(Coviello 2008)
• IGF-1
- ↑ se IGF-1
IGF 1 b
by androgens
d
(
(Bhasin
2001, Navarro 2002, Sheashaa
S
2005)
• SCF
- ↑ SCF in cultured BM stromal cells (Kim 2005) – synthetic
androgen
g oxymetholone
y
and 5β-dihydrotestosterone
β
y
(non-androgenic steroid)
Rationale for study
•
Androgens increase erythropoiesis however
mechanism is unclear
•
Most of the studies using animal and cell models are
more than 20yrs old and many inconclusive
•
-
EPO
widely
de y used to
o treat
ea a
anaemia
ae a
subset of patients hyporesponsive
some adverse findings in cancer patients
increased risk of cardiovascular disease in CRD
so alternatives to EPO desirable
Androgen action:
aromatase
Testosterone
estradiol
ER
5α-reductase
5α-dihydrotestosterone (DHT)
Androgen receptor (AR)
Androgen Receptor
T
T
T
T
T
T
AR
AR
nucleus
T
T
T
AR AR
T
AR AR
co-factors
ARE
cytoplasm
t l
Target gene
regulation
Experimental model –
Gl b l androgen
Global
d
receptor
t kknockout
k t (ARKO)
mouse model
•X chromosome
•Targeted exon 3 - 2nd zinc finger of DNA binding domain
•Natural deletion in humans causes complete AIS (Quigley et al 1992)
•Global ARKO males display murine AIS
•XY
XY genotype but female-like
female like phenotype
•Agenesis of seminal vesicles
•Internalised
Internalised testes
•Serum testosterone ↓80% cf wt males
WT male
WT female
global ARKO male
g
Notini AJ, Davey RA, McManus JF et al, J Mol Endo, 2005, 35:547-55
ARKO males vs wt littermates
(9wks)
Kidney mass
Bodyy mass
↓24% p<0.0001
35
30
500
↑8% p<0.05
25
↓30% p<0.001
400
300
g
mg
20
15
10
Need
p values!!!!!
200
100
5
0
0
Testes mass
200
Spleen/body mass as %
↓93 p<0.0001
0.7
↓66% p<0.001
↓39% p<0.01
0.6
mg
150
0.5
0.4
100
0.3
0.2
50
0.1
0
0
n=10-15/gp
wt male
ARKO male
wt female
Experimental design
• Baseline erythropoiesis in male ARKO mice and wt
litt
littermates
t
– blood, BM and spleen
– serum EPO,
EPO kidney EPO mRNA expression
• Phenylhydrazine
y y
treatment ((induce haemolytic
y anaemia))
• Androgen treatment – supraphysiological levels of
androgens
testosterone (6 weeks)
dihydrotestosterone
y
((DHT)) ((6,, 2 and 1 week))
via subcutaneous silastic tubing implants
- vehicle (empty tubing)
– blood,
bl d BM
BM, spleen
l
– serum EPO, kidney EPO expression
Baseline Erythropoiesis - Blood
No difference in RBC parameters between
untreated male ARKO mice and wt
controls at 6 or 9 weeks
(In a subset
(I
b
off 3 wt males
l and
d 3 ARKO
parameters were
littermates RBC p
decreased)
Baseline erythropoiesis –
Bone Marrow and Spleen
p
BM
140
↓15% p<0.05
120
↓32% p<0.01
4
10
100
%
80
60
40
CD71
20
0
Region
Spleen
1
2
3
4
0
10
0
10
160
↓32% p<0.05
140
120
R3
R2
R4
R3
R5
R4
1
10
2
10
FL2-H
3
10
Ter119
100
%
R6
R1
F 3
10
L
1
2
- 10
H
1
10
80
average of 3 expts
60
wt male
40
20
ARKO male
0
Region
1
2
3
4
n=6/gp
?Androgens increase proliferation of immature erythroid progenitors
Results from 3 expts: n=2, 1 and 3 per gp
?Androgens
increase promote differentiation
?skewed by n=3 gp where rbc parameters in arko lower than wt
?Androgens make immature progenitors available for EPO action
4
10
Serum EPO in 6 w.o. untreated ARKO
males vs wt littermates
wt male
300
ARKO male
250
200
pg/mL
wt female
↓30% p<0.05
150
p=0.053
100
50
0
n = 27
12
23
•No difference in serum EPO at 9 w.o.
•No difference in kidneyy EPO expression
p
at 9 w.o.
?AR regulation of EPO at 6 w.o.
Impact of AR KO on other lineages
in 9 w.o. mice
WBC
L
Lymphocytes
h
t
10
9
9
8
8
7
6
6
x 10(9)/L
x10(9)/L
7
5
4
5
4
3
3
2
2
1
1
0
0
N t hil
Neutrophils
1.8
1.6
1.4
n=14-16/gp
x10(9)/L
1.2
1
0.8
0.6
0.4
0.2
0
↓46 p<0.05
↓43 p<0.05
↑49% p<0.05
Impact of AR KO on other lineages
in 6 w.o. mice
L mphoc tes x10(9)/L
Lymphocytes
10(9)/L
WBC x10(9)/L
10(9)/L
↑58% p<0.001)
16
↑43% p
p<0.001
14
14
12
12
10
10
8
8
6
6
4
4
2
2
0
0
n=
28
↑67% p<0.0001
16
12
↑49% p<0.0001
p<0 0001
n=
27
N t hil x10(9)/L
Neutrophils
10(9)/L
28
12
27
Platelets
2000
0.6
05
0.5
↓17% p<0.01
↓22% p<0.0001
1500
0.4
0.3
1000
0.2
500
0.1
0
0
n=
28
12
27
n = 28
12
27
Validation of model:
+vehicle or +DHT
6 wk treatment
vehicle
DHT
kidney mass
600
testosterone
testes mass
↑25% p<0.0001 ↑65% p<0.0001
↓12% p<0.05
200
500
150
mg
mg
400
300
200
100
50
100
0
0
1 wk treatment DHT
kidney mass
500
seminal vesicle mass
↑31 p<0.001
300
250
400
200
mg
mg
300
200
↑49 p<0.0001
150
100
100
50
0
0
Androgen sensitive tissues respond to DHT in wt but NOT ARKO male mice
Phenylhydrazine induced haemolytic anaemia
- to assess reticulocyte response
phz - retics (%)
80
70
60
50
40
30
20
10
0
PHZ 60mg/kg days 0 and 1
arko m expressed as % wt m
4/
12
/2
00
7
4/
13
/2
00
7
4/
14
/2
00
7
4/
15
/2
00
7
4/
16
/2
00
7
4/
17
/2
00
7
4/
18
/2
00
7
4/
19
/2
00
7
%
wt male
ARKO male
n=
7
9
160
140
*
120
100
80
60
40
20
0
day 2
day 3
day 6
day 9
average of 3 expts
No difference in acute anaemia response at 9 w
w.o.
o
between male arko wt and mice
RBC parameters 6 wks post vehicle, testosterone or DHT
RBC
HCT
↑4% (p<0.05)
10
60
x10(12)
8
Hb
↑2.8% (p<0.05)
p=0.098
50
40
6
30
4
20
2
10
0
0
n = 13 11 10
160
7
↑3% (p<0
(p<0.05)
05)
9
6
9
5
11
n = 13
11 10
7
9
6
9
11
↑3 3% (p=0
↑3.3%
(p 0.051)
051)
p=0
p
0.06
06
Reticulocytes not increased in wt mice
140
120
100
g /L
5
80
+ vehicle
+ testosterone
+ DHT
60
40
20
0
↓
↓4%
(p=0.061)
n = 13 11 10
7
9
6
9
5
11
DHT increases RBC parameters in wt littermates
(3-4%, p<0.05) but NOT male ARKO mice →
ANDROGENS act through the AR
Serum EPO 6 wks post vehicle, testosterone
or DHT
350 ↓49% p<0.05
↓68% p
p<0.01
300
p=0.058
pg/mL
250
200
+ vehicle
+ testosterone
+ DHT
150
100
50
0
n=7
8
5
12
11
9
8
6
11
Kid
Kidney
EPO expression
i also
l d
decreased
d iin ttestosterone
t t
ttreated
t d wtt males
l
•Androgens do not increase serum EPO
•Serum EPO is decreased post testosterone but NOT DHT in both male
ARKO and wt mice (action likely via the estrogen receptor)
Ter119 pos/CD71hi cells in bone marrow
6 wks post vehicle or DHT by flow cytometry
(expressed as a % of male wt mice treated with vehicle):
↓25% p<0.0001
140
%
4
10
↑15% p<0.01
120
wt m + veh
100
wt m + dht
80
arko m + veh
60
arko m + dht
wt f + veh
40
wt f + dht
20
0
n=
10
6
5
3
5
6
CD71
R6
F 3
10
L
1
2
- 10
H
1
10
0
10
0
10
R
1
R3
R2
R4
R3
R5
R4
1
10
2
10
FL2 H
FL2-H
3
10
4
10
Ter119
?Androgens increase proliferation of immature erythroid progenitors in BM via the AR
Results averaged from 3 expts
Ter119 pos/CD71hi cells in spleen 6 wks post vehicle or DHT
by flow cytometry
(
(expressed
d as a % off wtt mice
i ttreated
t d with
ith vehicle):
hi l )
p=0.057
250
↑75% p<0.05
0 05
200
150
100
50
0
n = 10
300
250
6
5
3
↑107% pp=0.057
Results averaged from 3 expts
200
150
100
50
0
n=
5
6
?Androgens increase proliferation of immature erythroid progenitors in spleen
RBC parameters 2 wks post vehicle or DHT
RBC
HCT
↑7% (p<0.001)
↑6.2% (p<0.001)
wt m + vehicle
10
wt m + DHT
50
arko m + vehicle
40
6
arko m + DHT
4
wtt f + vehicle
hi l
0
n=4
6
7
9
7
wt f + DHT
9
↑7.2% (p<0.005)
30
20
10
0
n= 4
Hb
↑4.8% (p<0.05)
60
%
x10(12)
8
2
6
7
9
7
9
↑5.4% (p=0.078)
20
Reticulocytes not increased in wt mice
No increase in serum EPO
No increase in kidney EPO expression
15
g /d L
↑9% (p<0.001)
12
10
5
0
n=
4
6
7
9
7
9
DHT (2 wk treatment) increases
erythropoiesis via the AR
Absolute Reticulocytes 1 wk post vehicle or DHT
wt male + veh
n=
5
700
wt male + DHT
5
500
600
↑ 109 p<0.05
↑ 46% p<0.05
400
wt female + veh
4
wt female
f
+ DHT
5
300
200
100
0
No change in other RBC parameters
No increase in serum EPO
No increase in kidney EPO expression
Androgens increase erythropoiesis without increasing EPO
WBC x10(9)/L
post vehicle or DHT
1 week
12
↑59% p<0.01
10
2 weeks
k
8
12
6
10
4
8
2
6
0
n=
4
5
4
5
6 weeks
2
0
5
n= 4
6
7
9
7
9
12
10
↓
↓32%
p<0.01
8
↓ WBC attributed to lymphocytes
at 6 wks
6
4
2
0
n= 6
5
13
10
7
11
Lymphocytes x10(9)/L post vehicle
or DHT
1 week
8
↑44% p<0.05
7
6
2 weeks
5
10
4
9
3
8
2
7
6
1
5
0
n=
4
3
5
5
4
5
6 weeks
2
1
0
n= 4
6
7
9
7
9
9
↓27% p
p=0.076 ↓47% p<0.0001
8
7
6
5
Suppression of B lymphopoiesis
occurs by
b 6 weeks
k
4
3
2
1
0
n= 6
5
13
10
7
11
Neutrophils x10(9)/L post vehicle or
DHT
1 week
3
2.5
2 weeks
k
↑154% p<0.01
p<0 01
2
1.5
2.5
1
2
↑59% p<0.05
1.5
0.5
0
n=
1
5
4
5
6 weeks
0.5
0
5
n= 4
6
7
9
7
9
neutros +veh/DHT
2
↑63% p=0.065
?androgens mediate erythropoiesis by
acting on a precursor common
to neutrophils and erythrocytes
1.5
1
0.5
0
n= 6
5
13
10
7
11
Conclusions:
• Androgens mediate erythropoiesis via the androgen
receptor
• The mechanism of androgen mediated erythropoiesis is
not via EPO upregulation
• AR may have a role in regulation of EPO in young
mice??
• Androgens promote proliferation of immature erythroid
cells??
• ER mayy regulate
g
EPO??
Looking ahead
ahead…
Anaemia therapies
•
•
•
•
Androgens – unsuitable
SARMs
Molecular mechanisms?
Identification of pathways → new
therapeutics
Acknowledgements
Department
D
t
t off Medicine,
M di i
A
Austin
ti
Health
Bone M
B
Marrow R
Research
h
Laboratories, RMH
Jeffrey Zajac
Helen MacLean
Rachel Davey
Maria Chiu
Amanda Notini
David Curtis
Matthew McCormack