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Lunch & Science Journal Club as part of the Center SCOR Award

Lunch & Science Journal Club as part of the Center SCOR Award. July 10, 2013: “Progesterone Receptor (PR) Regulates Bone Growth, in Bone?” presented by Zhendong “Alex” Zhong, Ph.D. Peak bone mass (PBM) predicts osteoporosis. Peak bone mass (PBM). arthrolink.com. Estrogen deficiency.

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Lunch & Science Journal Club as part of the Center SCOR Award

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  1. Lunch & Science Journal Club as part of the Center SCOR Award July 10, 2013: “Progesterone Receptor (PR) Regulates Bone Growth, in Bone?” presented by Zhendong “Alex” Zhong, Ph.D.

  2. Peak bone mass (PBM) predicts osteoporosis Peak bone mass (PBM) arthrolink.com Estrogen deficiency

  3. Sex hormones for bone homeostasis Gonads Sex steroids Testis Ovary Increase Osteoblast proliferation and differentiation Androgens Estrogens apoptosis Activity & apoptosis osteoclast osteoblast ? Ovary or Adrenal glands Progesterone

  4. Progesterone affects other sex hormones profoundly Wikipedia

  5. Progesterone Receptor (PR) Progesterone • Female menstrual cycle (cyclical) • Pregnancy (high, supports gestation) • Embryogenesis • Others (bone?) Susan A. Leonhardt and  Dean P. Edwards 2002

  6. PR knockout (PRKO) mice show higher bone mass BV/TV • More dramatic HBM phenotype in females • Higher bone formation rate in females, lower bone resorption rate in males • PR antagonist RU486 also enhanced bone formation in normal female mice • Higher Osteoblast maturation-related genes, and lower apoptosis-related Yao, PLoSONE, 2010

  7. Further questions Which cell population is responsible? Which downstream signaling pathway of PR is responsible? What is the mechanism of the sex differences? The timing of PR knockout High bone mass in PRKO mice ? Hypothesis • PR regulates bone homeostasis within bone • The regulation has sex differences

  8. Specific aims Aim 1. To investigate the direct effects of PR deletion in osteoblasts on bone formation, and compare these effects in female and male mice Aim 2. To determine time-dependent and tissue-specific PR inhibition on bone turnover and bone mass accrual

  9. Bone Cell Differentiation Hematopoietic stem cell TRAP-Cre CtsK-Cre Mesenchymal Stem cell Adipocyte; myocyte; neuron Col10α1-Cre Osx1-Cre MSC Osteoclast Runx2+ ColXα1+ Sox9+ Col2α1+ Skeletal progenitor Sox9+ Runx2+ ?? Chondrocyte Hypertrophicchondrocyte Col2α1+ Runx2+ Osx1+ Oc+ CapG+ Dmp1+ SOST+ FGF23+ ORP150 Skeletal primordium Osteoblast progenitor Osteoblast precursor Osteoblast precursor Osteoblast Mineralizing osteocyte Mature osteocyte Prx1-Cre Dermo1-Cre Col2α1-Cre Oc-Cre Dmp1-Cre Sox9-Cre Col1α1-Cre (3.6kb) Osx1-Cre Col1α1-Cre (2.3kb) Mx1-Cre Figure adapted from Bonewald,2011, Vankoevering, 2008 and Rodda, S. J. 2006

  10. Generation of an inducible & bone-specific knockout of PR 1. Mx1-Cre Osteoprogenitors 2. Col1a1-CreERT2 Mature osteoblasts X PR-flox/flox Cre-Tg/+ X Tg/+, flox/+ Induce X pI-pCor Tamoxifen Tg/+, flox/flox PR is knocked out in “osteoprogenitors” or “mature osteoblasts” More tg/+, flox/flox +/+, flox/flox

  11. Mx1-Cre;PR-flox heterozygotes Tg, flox/+, Female pI-pC IP injection starts at 1 month of age Bone volume (white) / Total volume (BV/TV) 3mo, Female

  12. Challenges • To induce PR gene knockout after 1 month of age Col1a1-CreERT2 does not respond well to Tamoxifen induction after 2 weeks of age • How well is Cre activated in different compartments? Background activity? Specificities?

  13. Determine the knockout efficiency using mT/mG model “mT/mG” Col1a1-CreERT2 or Mx1-Cre × Mx1-Cre; mTmG mice Stop codon Stop codon tdTomato EGFP loxP loxP ATG Cell Stem Cell, 2012

  14. Mx1-Cre;mTmG mice, induced in vivo at 4 weeks of age Green: GFP /Mx1 activity Red: Tomato /all others Osteoclastic differentiation Blue: DAPI/nuclei Bright field No induction Induced in vivo Mx1-Cre; mTmG Col1-Cre; mTmG Bone marrow cells from the femurs, in vitro cultured for overnight

  15. Mx1-Cre;mTmG mice, induced in vivo at 4 weeks of age • Bone marrow stromal cells were differentiated into osteoblasts, for 7 days Non-induced Induced No differentiation 14d differentiation • stromal cells were differentiated into osteoblasts Mx1-Cre is active in a part of osteoprogenitors before induction; Mx1-Cre is harder to be activated in mineralized osteocytes?

  16. Ex vivo study on PR signaling control Induced (Mx1-Cre) Induced (Col1-Cre) Bone growth Mx1/Col1-Cre; PR-flox PR protein Osteoblast differentiation, TGFβ/IHH/Wnt signaling changes

  17. Summary • Generated conditional/inducible PR knockout mice, measuring bone mass changes post induction • Investigating the timing and dosage for TM/pl-pC induction Next steps • Measure bone mass in homozygotes • New models: Prx1-Cre/ Oc-Cre • Study Wnt/IHH/TGF pathways

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