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 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 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


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).


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 phenotpye in Bone-fat phenotpye in POHPOH

• Osteoblast differentiation and HO



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


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


*

**

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


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


0

0.5

1

1.5

2

2.5

3

3.5

0 9 17 20

Gnas+/+

Gnas+/-

A


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


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





• Adipocyte differentiation and leaness


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)


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


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.


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


WT Gsα+/p- WT Gsα+/p-

Fat Pad(subcutaneous)

WAT (abdominal)

BAT(interscapular)

Inactivating Gsα mutation reduces adipogenic tissues in vivo II


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.


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

****


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


WT Gsα+/p-


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






• 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

robert j. pignolo, m.d., ph.d

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