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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 differentiation and HO

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Robert J. Pignolo, M.D., Ph.D.

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  1. Bone-fat reciprocity in progressive osseous heteroplasia (POH)Reciprocità tra osso e tessuto adiposo nella POH Robert J. Pignolo, M.D., Ph.D.

  2. 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

  3. 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

  4. 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]

  5. Diagnosis Superficial Deep > 2 AHO PTH n a b c HO HO Features Resistance POH 52 + + - - d POH/AHO 6 + + + - d d POH/PHP1a/1c 5 + + + + d Oste oma cutis 26 + - - - d d AHO 10 + - + - d e d PHP 1a/1c 12 + - + + 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. Clinical characteristics of POH and other GNAS-based disorders of HO Adegbite et al Am J Med Gen (2008)

  6. Progression of HO in POH 18 months 30 months 8 years Kaplan, F. S. et al. J. Bone Joint Surg. 76, 425–436 (1994)

  7. Spectrum of POH and other GNAS-based conditions of HO Adegbite et al Am J Med Gen (2008)

  8. Low birth weight in patients with POH 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).

  9. Obesity and other AHO features among patients with GNAS-based disorders of HO Adegbite et al Am J Med Gen (2008)

  10. Bone-fat reciprocity in progressive osseous heteroplasia (POH) • Bone-fat phenotpye in POH • Osteoblast differentiation and HO • Adipocyte differentiation and leaness • Mechanisms of bone-fat reciprocity

  11. GNAS encodes multiple transcripts Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)

  12. Bone formation in POH Shore EM & Kaplan FS Nat Rev Rheumatol 6: 518-527 (2010)

  13. Gsα XLαs NESP No 1o Ab Bone Fat Expression of Gnas protein products in bone and fat Pignolo et al, Submitted (2011)

  14. * * * Expression of Gnas transcripts in adipocyte soft tissue stromal cells (STSCs) Pignolo et al, JBMR 25: 2647-55 (2011)

  15. A WT 749 WT 750 WT 756 WT 763 B KO 765 KO 752 KO 754 KO 764  Accelerated osteoblast differentiation in STSCs from Gnas+/- mice Pignolo et al, JBMR 25: 2647-55 (2011)

  16. A   Relative ALP mRNA expression Gnas+/+ Gnas+/+ Gnas+/+ 3.5 3 Gnas+/- Gnas+/- Gnas+/- 2.5 0 2 17 20 9 1.5 Osteoblast differentiation (days) 1 B 0.5 0  Relative OP mRNA expression 0 9 17 20 3 Osteoblast differentiation (days) 2.5 C 2 1.5 1  0.5 Relative OC mRNA expression 0 0 9 17 20 Osteoblast differentiation (days) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Expression of osteoblast markers in adipose-derived stem cells Pignolo et al, JBMR 25: 2647-55 (2011)

  17. A F C B F B D Gnas+/- mice develop HO Pignolo et al, JBMR 25: 2647-55 (2011)

  18. Bone-fat reciprocity in progressive osseous heteroplasia (POH) • Bone-fat phenotpyein POH • Osteoblast differentiation and HO • Adipocyte differentiation and leaness • Mechanisms of bone-fat reciprocity

  19. Paternally-inherited Gsα mutation impairs adipogenesis in vitro ** A B WT Gsα+/p- Relative amounts of Oil Red O (day10) WT Gsα+/p- Adipogenesis WT Gsα+/p- C C/EBPβ C/EBPα PPARγ aP2 Relative RNA expression * * * * [ [ [ [ C 1 3 7 C 1 3 7 C 1 3 7 C 1 3 7 Adipogenic differentiation (days) Lui et al, submitted (2012)

  20. * * * Expression of Gnas transcripts during adipocyte differentiation WT Gsα+/p- A ** Relative Gsα RNA expression 0 1 3 7 B Relative XLαs RNA expression 0 1 3 7 C Relative Nesp RNA expression 0 1 3 7 Adipogenic differentiation (days) Lui et al, submitted (2012)

  21. Lui et al, submitted (2012)

  22. Inactivating Gsα mutation reduces adipogenic tissues in vivo I Lui et al, submitted (2012)

  23. Inactivating Gsα mutation reduces adipogenic tissues in vivo II WT Gsα+/p- WT Gsα+/p- Fat Pad (subcutaneous) WAT (abdominal) BAT (interscapular) Lui et al, submitted (2012)

  24. Lui et al, submitted (2012)

  25. Expression of adipose markers in adipose tissues WT Gsα+/p- Fat Pad Relative RNA expression Gsα αP2 PPARγ Leptin ** ** White fat tissue (WAT) Relative RNA expression Gsα αP2 PPARγ Leptin Brown fat tissue (BAT) * * * * * * Relative RNA expression Gsα αP2 PPARγ Leptin UCP1 Lui et al, submitted (2012)

  26. Adenylyl cyclase activation rescues the adipogenic impairment of Gsα+/p- adipose-derived stem cells A day 1 Differentiation day 10 B forskolin WT Gsα+/p- day 1-2 forskolin day 5-6 Relative amounts Oil Red O (day 10) s -- + day 1-2 -- + day 1-2 + day 5-6 Forskolin: Adipogenic Induction: -- -- + + + * * Lui et al, submitted (2012)

  27. Paternally-inherited Gsα mutation potentiates osteogenesis during adipogenic induction WT Gsα+/p- A Relative Msx2 RNA expression B Relative Runx2 RNA expression 0 1 3 7 * * * * C Relative Osteocalcin RNA expression 0 1 3 7 Adipogenic differentiation (days) Lui et al, submitted (2012)

  28. Bone-fat reciprocity in progressive osseous heteroplasia (POH) • Bone-fat phenotpyein POH • Osteoblast differentiation and HO • Adipocyte differentiation and leaness • Mechanisms of bone-fat reciprocity

  29. Gsα XLαs ↓Gsα ↓XLαs ? Regulation of bone-fat reciprocity in soft tissue by GNAS/Gnas ↓ Osteogenic genes Gnas+/+ (wild-type) ↑↑↑ cAMP HO ↑ Adipogenic genes ↓Gsα Superficial HO Gnas+/- (exon 1) ↑↑cAMP XLαs ↑ Osteogenic genes ↓ Adipogenic genes Leaness POH ↑ cAMP Progressive HO

  30. 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

  31. Laboratory NichelleAdegbite Jan-Jan Lui Elizabeth Russell Alec Richardson MeiqiXu 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 Acknowledgements

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