Robert J. Pignolo, M.D., Ph.D.
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Transcript of Robert J. Pignolo, M.D., Ph.D.
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 Osteoblast differentiation and HOdifferentiation and HO
• Adipocyte Adipocyte differentiation and differentiation and leanessleaness
• Mechanisms of bone-Mechanisms of bone-fat reciprocityfat reciprocity
Bone-fat reciprocity in progressive osseous heteroplasia (POH)
• Bone-fat phenotype in POH
• Osteoblast differentiation and HO
• Adipocyte differentiation and leaness
• Mechanisms of bone-fat 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 G-stimulatory protein of adenylyl cyclase [Shore et al., 2002]
Adegbite et al Am J Med Gen (2008)
Diagnosis n Superficial HO
Deep HOa
> 2 AHO Featuresb
PTH Resistancec
POH 52 + + - - POH/AHO 6 + + +d - POH/PHP1a/1c 5 + + +d +d Osteoma cutis 26 + -d - - AHO 10 + -d +d - PHP 1a/1c 12 + -d +e +d
Clinical characteristics of POH and other GNAS-based disorders of HO
All (+) or (no (-) patients within the diagnostic category displayed the indicated characteristic.
aDeep HO refers to the extension of superficial (dermal) HO to deep tissues.
bAHO 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.
Progression of HO in POH
Kaplan, F. S. et al. J. Bone Joint Surg. 76, 425–436 (1994)
18 months 30 months 8 years
Spectrum of POH and other GNAS-based 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 hadmeasurements at or below the fifth percentile. Birth weights are plotted onto normative growthcharts 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 Stat2002;246:1—190).
Adegbite et al Am J Med Gen (2008)
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/PHP1a/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
Bone-fat reciprocity in progressive osseous heteroplasia (POH)
• Bone-fat phenotpye in Bone-fat phenotpye in POHPOH
• Osteoblast differentiation and HO
• Adipocyte Adipocyte differentiation and differentiation and leanessleaness
• Mechanisms of bone-Mechanisms of bone-fat reciprocityfat 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)
Pignolo et al, Submitted (2011)
Expression of Gnas protein products in bone and fat
Bone
Fat
No 1o AbXLαsGsα NESP
0
0.5
1
1.5
2
2.5
Wild-type Gnas+/-
Genotype
Rel
ativ
e G
sa E
xpre
ssio
n i
n S
TS
C- Ob Differentiation + Ob Differentiation
0
0.2
0.4
0.6
0.8
1
1.2
Wild-type Gnas+/-
Genotype
Rel
ativ
e 1A
Exp
ress
ion
in
ST
SC
- Ob Differentiation + Ob Differentiation
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Wild-type Gnas+/-
Genotype
Rel
ativ
e N
esp
Exp
ress
ion
in
ST
SC
- Ob Differentiation + Ob Differentiation
0
0.2
0.4
0.6
0.8
1
1.2
Wild-type Gnas+/-
Genotype
Rel
ativ
e X
La
s E
xpre
ssio
n i
n S
TS
C
- Ob Differentiation + Ob Differentiation
*
**
Expression of Gnas transcripts in adipocyte soft tissue stromal cells (STSCs)
Pignolo et al, JBMR 25: 2647-55 (2011)
KO 754
WT 750
KO 764
WT 756
KO 765
WT 763
KO 752
WT 749A
0
1
2
3
4
5
6
Day 9 Day 17 Day 20
Time (days)
Ali
zari
n R
ed S
/ug
pro
tein
WT
mut
B
Accelerated osteoblast differentiation in STSCs from Gnas+/- mice
Pignolo et al, JBMR 25: 2647-55 (2011)
Expression of osteoblast markers in adipose-derived stem cells
Osteoblast differentiation (days)
Rel
ativ
e O
P m
RN
A e
xpre
ssio
n
0
0.5
1
1.5
2
2.5
3
0 9 17 20
Gnas+/+
Gnas+/-
B
Rel
ativ
e A
LP
mR
NA
ex
pre
ssio
n
Osteoblast differentiation (days)
0
0.5
1
1.5
2
2.5
3
3.5
0 9 17 20
Gnas+/+
Gnas+/-
A
Osteoblast differentiation (days)
Rel
ativ
e O
C m
RN
Aex
pre
ssio
n
0
0.1
0.2
0.30.4
0.5
0.6
0.7
0 9 17 20
Gnas+/+
Gnas+/-
C
Pignolo et al, JBMR 25: 2647-55 (2011)
A
B
0
2
4
6
8
10
12
14
3 6 9 12 24
Average Age (Months)
Nu
mb
er o
f G
nas
+/- M
ice
HO
No HO
D
C
B
F
F
Gnas+/- mice develop HO
Pignolo et al, JBMR 25: 2647-55 (2011)
Bone-fat reciprocity in progressive osseous heteroplasia (POH)
• Bone-fat phenotpye in Bone-fat phenotpye in POHPOH
• Osteoblast Osteoblast differentiation and HOdifferentiation and HO
• Adipocyte differentiation and leaness
• Mechanisms of bone-Mechanisms of bone-fat reciprocityfat reciprocity
Paternally-inherited Gsα mutation impairs adipogenesis in vitro
A WT
Gsα+/p-
Rel
ativ
e am
ou
nts
of
Oil
Red
O
(day
10)
WT Gsα+/p-
Adipogenesis
**
B
Adipogenic differentiation (days)
WT Gsα+/p-
Re
lativ
e R
NA
exp
ress
ion
C/EBPβ
[*
C 3 71
C/EBPα [*
C 1 3 7
PPARγ [*
C 1 3 7
aP2
[*
C 1 3 7
C
Lui et al, submitted (2012)
Adipogenic differentiation (days)
WT Gsα+/p-
A
Rel
ativ
e G
sα
RN
A e
xpre
ssio
n
0 1 3 7
**
B
Rel
ativ
e X
Lα
s R
NA
exp
ress
ion
1 3 70
*
C
Rel
ativ
e N
esp
R
NA
exp
ress
ion
0 1 3 7
* *
Expression of Gnas transcripts during adipocyte differentiation
Lui et al, submitted (2012)
Table 1. Anthromorphometric and gross adipose tissue measurements
Genotype Total mouse weight (g)
Length (cm)
BMI (g/cm2)
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 < 0.001, 2-sided t-test Gsa+/p-
(KO) versus WT mice; ns, not significant.
Lui et al, submitted (2012)
Inactivating Gsα mutation reduces adipogenic tissues in vivo I
BAT
WAT
FP
Wild-type Gsα+/p-
A
B
WAT
FP FP
BAT
FP
BA
BAT
WAT
FP
Wild-type Gsα+/p-
A
B
WAT
FP FP
BAT
FP
BA
Lui et al, submitted (2012)
WT Gsα+/p- WT Gsα+/p-
Fat Pad(subcutaneous)
WAT (abdominal)
BAT(interscapular)
Inactivating Gsα mutation reduces adipogenic tissues in vivo II
Lui et al, submitted (2012)
Table 2. Histomorphometric analysis of adipose tissues
N
(# cells counted) Mean cell size
(μm2) % stroma
Adipocyte number/
10000 μm2
FP WT 254 1704±425 28±3.8 4±1.1
KO 544 642±87.9*** 36±3.3** 10±1.8***
WAT WT 212 2853±512 31±3.6 2.4±0.53
KO 635 1883±332*** 31±5.8 3.7±0.78***
BAT WT 784 319±53 44±7.2 17±2.8
KO 435 259±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 < 0.05, ** p < 0.01, *** p < 0.001, 2-sided t-test Gsa+/p-
(KO) versus WT mice.
Lui et al, submitted (2012)
Expression of adipose markers in adipose tissues
WT Gsα+/p-R
elat
ive
RN
A
expr
essi
on
Brown fat tissue (BAT)
Gsα αP2 PPARγ Leptin UCP1
* *
Rel
ativ
e R
NA
ex
pres
sion
White fat tissue (WAT)
Gsα αP2 PPARγ Leptin
* ***
Rel
ativ
e R
NA
ex
pres
sion
Fat Pad
Gsα αP2 PPARγ Leptin
****
Lui et al, submitted (2012)
B
s
WT Gsα+/p-
* *
Rel
ativ
e a
mou
nts
Oil
Red
O
(da
y 10
)
-- + day 1-2 -- + day 1-2 + day 5-6 Forskolin:
-- -- + + +AdipogenicInduction:
Differentiation
day 1-2forskolin
forskolin
A
day 1 day 10
day 5-6
Adenylyl cyclase activation rescues the adipogenic impairment of Gsα+/p- adipose-derived stem cells
Lui et al, submitted (2012)
WT Gsα+/p-
Adipogenic differentiation (days)
C*
Re
lativ
e O
ste
oca
lcin
R
NA
exp
ress
ion
0 1 3 7
A
Re
lativ
e M
sx2
R
NA
exp
ress
ion *
*
B
Re
lativ
e R
un
x2
RN
A e
xpre
ssio
n
0 1 3 7
*
Paternally-inherited Gsα mutation potentiates osteogenesis during adipogenic induction
Lui et al, submitted (2012)
Bone-fat reciprocity in progressive osseous heteroplasia (POH)
• Bone-fat phenotpye in Bone-fat phenotpye in POHPOH
• Osteoblast Osteoblast differentiation and HOdifferentiation and HO
• Adipocyte Adipocyte differentiation and differentiation and leanessleaness
• Mechanisms of bone-fat reciprocity
Gnas+/-
(exon 1)
↓Gsα
XLαs↑↑cAMP
Regulation of bone-fat reciprocity in soft tissue by GNAS/Gnas
Gnas+/+
(wild-type)
Gsα
XLαs↑↑↑ cAMP HO
POH↓Gsα
↓XLαs↑ cAMP?
Superficial HO
Progressive HO
Leaness↑ Osteogenic genes↓ Adipogenic genes
↓ Osteogenic genes
↑ Adipogenic genes
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