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DIVISION OF
RHEUMATOLOGY
Arginine and the Metabolic Control of Osteoclast Generation
Brunner JS1, Hofmann M2, Saferding V2, Paar H1, Chen L3, Cheng P3, Schabbauer G1, Blüml S2
1Medical
University Vienna, Institute for Physiology, Vienna, Austria; 2Medical University Vienna, Dpt. of Rheumatology, Vienna, Austria; 3Bio Cancer Treatment International Ltd., Hong Kong, China
Background:
L-arginine is a semi-essential amino acid, known for its prominent role in cellular metabolism.
M-CSF
M-CSF-R
RANKL
Availability and catabolism of arginine have been implicated with immune cell biology,
RANK
skewing inflammatory responses within myeloid cells in a pro- or anti-inflammatory manner.
PI3K
While the role of arginine within certain myeloid lineages such as macrophages is well
MAPK
appreciated, its role within osteoclasts is relatively unknown.
AKT
NF-κB
Methods:
Arginine
S6K
?
?
mTOR
We analyzed osteoclastogenesis of C57BL/6J or BALB/c wildtype cells in vitro in the
NFATc1
presence and absence of recombinant arginase I (recARG1) and its inhibitor nor-NOHA.
This approach was complemented via qPCR analysis of relevant marker genes. We
Arginine’s central role in the M1-M2 macrophage polarization paradigm and its
further investigated the potential of the enzyme to induce cell death via flow cytometry
proposed influence on osteoclast differentiation
analysis of 7-AAD and Annexin V.
The Importance of L-Arginine in Osteoclastogenesis
A
B
C
7-AAD+ Annexin V+
Arginase I
Control
100
*
300 ng/ml
recARG1
50
**
-15
ay
D
D
ay
7
3
Day 7: Osteoclast
20
0
Co
nt
ro
l
10
0
30
0
1
00
0
0
70
30
10
1 000 ng/ml
recARG1
n.d.
80
HS
-10
100
l
-5
90
Co
nt
ro
+M-CSF
+RANKL
0
% of parent
Day 3: Osteoclast
Precursor
Number of Osteoclasts
+M-CSF
Fold change
norm. to HPRT
150
00
0
10
00
0
5
1
Day 0: Myeloid
Progenitor
B
10
0
A
recARG1 [ng/ml]
recARG1 [ng/ml]
Figure 2: Osteoclast assay of differentiated wildtype bone marrow derived macrophages treated with
Figure 1: Expression levels of arginase I after induction of
recARG1. A) Counted osteoclasts containing at least 3 nuclei. B) Representative microscope pictures taken from the same
osteoclastogenesis. A) Schematic of an in vitro osteoclast assay. B)
experiment, stained for tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. C) Flow cytometry analysis of 7-AAD
Messenger RNA levels of arginase I decrease after addition of RANKL on day
and Annexin V double positive cells, harvested on day 4 after differentiation, one day after incubation with RANKL and recARG1
3 of osteoclast differentiation.
(HS: Heat shock).
Results:
We incubated day 3 osteoclast precursors with recARG1
B
A
Control
300 µM nor-NOHA
Denatured
80
60
40
20
n.d.
100
150
100
50
0
Depl. Medium
Black 6
50
****
n.d.
Depl. Medium
Balb/c
5
4
3
2
1
0
n.d.
Depl. Medium
Black 6
**
n.d.
Depl. Medium
differentiation
assay.
Strikingly,
combined treatment of recARG1 with the arginase I
specific inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) or
25
by denaturing the enzyme, confirming that the enzymatic
**
activity of recARG1 is necessary for the inhibitory effect.
We depleted osteoclast generation cell culture medium of
C
200
TRAP
added only in the M-CSF-dependent first phase of the
osteoclastogenesis could be completely restored upon
Fold change normalized to HPRT
Number of Osteoclasts
250
Fold change normalized to HPRT
C
recARG1 did not affect the generation of osteoclasts when
osteoclast
75
0
1 µg/ml
recARG1
n.d.
Control
1 µg/ml recARG1
on
tr
ol
D
ay
03
D
ay
36
0
abolished the formation of mature osteoclasts. Further,
125
Number of Osteoclasts
Number of Osteoclasts
100
and observed that addition of 1 000 ng/ml recARG1
L-arginine using recARG1, then blocked the enzyme using
Cathepsin K
5
**
4
Control
400 µM L-Arg
nor-NOHA and found osteoclastogenesis to be completely
blocked using this “depleted” medium. Osteoclastogenesis
3
could be restored by reconstituting the depleted medium
2
with L-arginine.
1
0
Balb/c
n.d.
Depl. Medium
Black 6
**
n.d.
Depl. Medium
Balb/c
Conclusion:
We propose that the essential amino acid L-
Figure 3: Effects of recARG1 on osteoclastogenesis are linked to RANKL signaling and its enzymatic activity. The
effects can be rescued by L-arginine resupplementation. A) Inhibition of the enzymatic activity via denaturation or inhibition of the
arginine is critical for the development of
enzyme rescues osteoclastogenesis. B) Incubation with 1 µg/ml recARG1 for the first three days during MCSF incubation does not influence later
osteoclasts
osteoclast formation. C) Counted osteoclasts containing at least 3 nuclei and fold induction in developing osteoclasts incubated with depleted
hypothesize that its abundance might influence
medium or resupplied medium after 7 days of differentiation shown for TRAP and cathepsin K.
from
myeloid
precursors
development and severity of osteoporosis.
and