Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
Reciprocità tra osso e tessuto adiposo
nella POH
Robert J. Pignolo, M.D., Ph.D.
Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
• Bone-fat phenotype in
POH
• Osteoblast
differentiation and HO
• Adipocyte
differentiation and
leaness
• Mechanisms of bonefat reciprocity
Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
• Bone-fat phenotype in
POH
• Osteoblast
differentiation and HO
• Adipocyte
differentiation and
leaness
• Mechanisms of bonefat reciprocity
Progressive osseous heteroplasia
(POH; OMIM #166350)
• Rare genetic condition of progressive
heterotopic bone formation
• Defined clinically by cutaneous ossification that
progresses to involve deep connective tissues
including skeletal muscle and fascia [Kaplan et
al., 1994]
• Most cases are caused by heterozygous
inactivating germline mutations in the GNAS
gene that encodes the alpha subunit of the Gstimulatory protein of adenylyl cyclase [Shore et
al., 2002]
Clinical characteristics of POH and
other GNAS-based disorders of HO
Diagnosis
n
POH
POH/AHO
POH/PHP1a/1c
Osteoma cutis
AHO
PHP 1a/1c
52
6
5
26
10
12
Superficial
HO
+
+
+
+
+
+
Deep
HOa
+
+
+
-d
-d
-d
> 2 AHO
PTH
b
Features Resistancec
d
+
d
+
+d
d
+
e
+
+d
All (+) or (no (-) patients within the diagnostic category displayed the indicated characteristic.
aDeep
bAHO
HO refers to the extension of superficial (dermal) HO to deep tissues.
features included: short statue, obesity, round face, brachydactaly, neurobehavioral abnormalities.
The presence of heterotopic ossification was common to all presentations and excluded here.
cAn
endocrine evaluation included a survey of calcium, phosphorus, TSH, and intact PTH blood levels.
Two POH patients had endocrine abnormalities: one had a high TSH, and another had high calcium and phosphate levels.
One POH/AHO patient had a high phosphate level.
All POH/PHP 1a/1c patients had PTH resistance or PTH and thyroid hormone resistance.
Differences found to be statistically significant when compared to POH are d p < 0.0001, e p = 0,0002.
Adegbite et al Am J Med Gen (2008)
Progression of HO in POH
18 months
Kaplan, F. S. et al. J. Bone Joint Surg. 76, 425–436 (1994)
30 months
8 years
Spectrum of POH and other GNASbased conditions of HO
Adegbite et al Am J Med Gen (2008)
Low birth weight in patients with
POH
Shore, Supplemental Figure 1
Shown are birth weights from 13 patients with POH. Three of 4 males and 7 of 9 females had
measurements at or below the fifth percentile. Birth weights are plotted onto normative growth
charts for sex-matched cohorts born in United States (Kuczmarski RJ, Ogden CL, Guo SS, et al.
2000 CDC growth charts for the United States: methods and development. Vital Health Stat
2002;246:1—190).
Obesity and other AHO features among
patients with GNAS-based disorders of HO
Diagnosis
Average no.
AHO
features per
patient
(+/- SD)
Short
Stature
(%)
Obesity
(%)
Round face
(%)
Brachydactaly
(%)
Mental
retardation
(%)
POH
0.31 (0.61)
7.7
0.0
3.8
15.4
3.8
POH/AHO
2.7 (0.5)
66.7
33.3
66.7
83.3
16.6
POH/PHP1
a/1c
2.2 (1.5)
80
40.0
20.0
40.0
40.0
Osteoma
cutis
0.0 (0.0)
0.0
0.0
0.0
0.0
0.0
AHO
2.7 (1.9)
50.0
40.0
50.0
80.0
20.0
PHP1a/1c
2.6 (1.4)
50.0
58.3
66.7
66.7
8.3
Adegbite et al Am J Med Gen (2008)
Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
• Bone-fat phenotpye in
POH
• Osteoblast
differentiation and HO
• Adipocyte
differentiation and
leaness
• Mechanisms of bonefat reciprocity
GNAS encodes multiple transcripts
Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)
Bone formation in POH
Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)
Expression of Gnas protein
products in bone and fat
Gsα
Bone
Fat
Pignolo et al, Submitted (2011)
XLαs
NESP
No 1o Ab
Expression of Gnas transcripts in adipocyte
soft tissue stromal cells (STSCs)
- Ob Differentiation
+ Ob Differentiation
- Ob Differentiation
1.2
Relative 1A Expression in STSC
Relative G sa Expression in STSC
2.5
2
1.5
1
0.5
0
1
*
0.8
0.6
0.4
0.2
0
Gnas+/-
Wild-type
- Ob Differentiation
Genotype
+ Ob Differentiation
- Ob Differentiation
0.7
1.2
*
Relative XL a s Expression in STSC
Relative Nesp Expression in STSC
Gnas+/-
Wild-type
Genotype
0.6
+ Ob Differentiation
0.5
0.4
0.3
0.2
0.1
0
+ Ob Differentiation
*
1
0.8
0.6
0.4
0.2
0
Gnas+/-
Wild-type
Genotype
Pignolo et al, JBMR 25: 2647-55 (2011)
Gnas+/-
Wild-type
Genotype
Accelerated osteoblast differentiation in
STSCs from Gnas+/- mice
A
B
WT 749
WT 750
WT 756
WT 763
KO 752
KO 754
KO 764
KO 765
6

Alizarin Red S/ug protein
5
4
WT
3
mut
2
1
0
Day 9
Day 17
Time (days)
Pignolo et al, JBMR 25: 2647-55 (2011)
Day 20
Expression of osteoblast markers in
adipose-derived stem cells
Relative ALP mRNA
expression
A
B
3.5
3
2.5
2
1.5
1
0.5
0
Relative OP mRNA
expression
3
2.5
2

Gnas+/+
Gnas+/-

0
17
20
9
Osteoblast differentiation (days)
Gnas+/+
Gnas+/-

1.5
1
0.5
0
0
9
17
20
Osteoblast differentiation (days)
Relative OC mRNA
expression
C
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Gnas+/+
Gnas+/-

0
9
17
20
Osteoblast differentiation (days)
Pignolo et al, JBMR 25: 2647-55 (2011)
Gnas+/- mice develop HO
A
C
F
B
F
B
14
D
Number of Gnas +/- Mice
12
10
8
HO
No HO
6
4
2
0
3
6
9
12
Average Age (Months)
Pignolo et al, JBMR 25: 2647-55 (2011)
24
Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
• Bone-fat phenotpye in
POH
• Osteoblast
differentiation and HO
• Adipocyte
differentiation and
leaness
• Mechanisms of bonefat reciprocity
Paternally-inherited Gsα mutation impairs
adipogenesis in vitro
A
B
Gsα+/p-
Relative amounts of Oil Red O
(day10)
WT
**
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Gsα+/p-
WT
Adipogenesis
WT
*
30
4
*
*
aP2
1000
25
25
3
PPARγ
30
[
C/EBPα
35
Gsα+/p-
[
C/EBPβ
[
5
500
20
20
2
*
[
Relative RNA expression
C
1
15
10
5
0
0
C
1
Lui et al, submitted (2012)
3
7
15
0
10
40
5
20
0
0
C
1
3
7
C
1
Adipogenic differentiation (days)
3
7
C
1
3
7
Expression of Gnas transcripts during adipocyte differentiation
WT
A
**
Relative Gsα
RNA expression
1.5
1.0
0.5
0.0
0
B
Relative XLαs
RNA expression
1.5
1
3
7
*
1.0
0.5
0.0
C
0
1
*
Relative Nesp
RNA expression
Gsα+/p-
3
7
*
3.0
2.0
1.0
0.0
0
Lui et al, submitted (2012)
1
3
Adipogenic differentiation (days)
7
Table 1. Anthromorphometric and gross adipose tissue measurements
Genotype
Total mouse
weight (g)
Length
(cm)
BMI
2
(g/cm )
Fat Pads
(g)
WAT
(g)
BAT
(g)
WT
(n=27)
36.91±1.98
9.55±0.24
0.41±0.02
0.88±0.24
0.87±0.22
0.20±0.03
KO
(n=16)
29.66±1.25***
8.94±0.24***
0.37±0.03 (ns)
0.44±0.08***
0.35±0.08***
0.11±0.03***
All values are average ± standard deviation. WAT, white adipose tissue; BAT, brown adipose tissue.
+/p*** p < 0.001, 2-sided t-test Gsa (KO) versus WT mice; ns, not significant.
Lui et al, submitted (2012)
BAT
WAT adipogenic
Inactivating Gsα mutation reduces
tissues in vivo I
FP FP
B
Wild-type
BAT
FP
BAT
WAT
BAT
WAT
FP FP
FP
Lui et al, submitted (2012)
+/p-
+/p-
Gsα
Inactivating Gsα mutation reduces
adipogenic tissues in vivo II
WT
Fat Pad
(subcutaneous)
WAT
(abdominal)
BAT
(interscapular)
Lui et al, submitted (2012)
Gsα+/p-
WT
Gsα+/p-
Table 2. Histomorphometric analysis of adipose tissues
N
(# cells counted)
FP
WAT
BAT
WT
254
KO
544
WT
212
KO
635
WT
784
KO
435
Mean cell size
2
(μm )
1704±425
642±87.9***
% stroma
28±3.8
Adipocyte
number/
2
10000 μm
4±1.1
36±3.3**
10±1.8***
2853±512
31±3.6
2.4±0.53
1883±332***
31±5.8
3.7±0.78***
319±53
44±7.2
17±2.8
259±25.8*
45±5.4
21±3.3*
All values are average ± standard deviation. FP, fat pads; WAT, white adipose tissue; BAT, brown adipose tissue.
+/p* p < 0.05, ** p < 0.01, *** p < 0.001, 2-sided t-test Gsa (KO) versus WT mice.
Lui et al, submitted (2012)
Expression of adipose markers in adipose tissues
Gsα+/p-
WT
Relative RNA
expression
Fat Pad
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
**
Gsα
αP2
*
*
PPARγ
Leptin
Relative RNA
expression
White fat tissue (WAT)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
**
Gsα
αP2
*
*
PPARγ
Leptin
Relative RNA
expression
Brown fat tissue (BAT)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
*
Gsα
αP2
*
PPARγ
Leptin
UCP1
Lui et al, submitted (2012)
Adenylyl cyclase activation rescues the adipogenic
impairment of Gsα+/p- adipose-derived stem cells
A
day 1
day 10
Differentiation
forskolin
day 1-2
forskolin
day 5-6
Relative amounts Oil Red
O (day 10)
B
*
0.50
WT
*
Gsα+/p-
0.40
0.30
s
0.20
0.10
0.00
Forskolin:
--
+ day 1-2
--
+ day 1-2
+ day 5-6
Adipogenic
Induction:
--
--
+
+
+
Lui et al, submitted (2012)
Paternally-inherited Gsα mutation potentiates osteogenesis
during adipogenic induction
WT
A
Gsα+/p-
Relative Msx2
RNA expression
5
*
4
*
3
2
1
0
0
1
3
7
Relative Runx2
RNA expression
B
*
0
Relative Osteocalcin
RNA expression
C
Lui et al, submitted (2012)
1
3
16
7
*
12
8
4
0
0
1
3
Adipogenic differentiation (days)
7
Bone-fat reciprocity in progressive
osseous heteroplasia (POH)
• Bone-fat phenotpye in
POH
• Osteoblast
differentiation and HO
• Adipocyte
differentiation and
leaness
• Mechanisms of bonefat reciprocity
Regulation of bone-fat reciprocity in
soft tissue by GNAS/Gnas
Gnas+/+
(wild-type)
Gsα
Gnas+/(exon 1)
↓Gsα
POH
XLαs
XLαs
?
↓Gsα
↓XLαs
↑↑↑ cAMP
↓ Osteogenic genes
↑ Adipogenic genes
HO
Superficial HO
↑↑cAMP
↑ Osteogenic genes
↓ Adipogenic genes
Leaness
↑ cAMP
Progressive HO
Summary
• Unlike other GNAS-based disorders of HO, POH
occurs in the absence of obesity and multiple
other features of AHO or hormone resistance
• Impaired bone-fat reciprocity in soft tissue
promotes osteogenesis and antagonizes
adipogenesis in POH
• GNAS is a key factor which regulates cell fate
decisions in adipose-derived stem cells
Acknowledgements
• Laboratory
–
–
–
–
–
–
Nichelle Adegbite
Jan-Jan Lui
Elizabeth Russell
Alec Richardson
Meiqi Xu
Josef Kaplan
• Collaborators
– Eileen Shore
– Fred Kaplan
• Funding
– National Institutes of Health
– John A. Hartford
Foundation
– International Progressive
Osseous Heteroplasia
Association
– Italian Progressive
Osseous Heteroplasia
Association
– Ian Cali Fund
– University of Pennsylvania
Center for Research in
FOP & Related Disorders