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 arthrolink.com Peak bone mass (PBM) Estrogen deficiency

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

Progesterone affects other sex hormones profoundly Wikipedia

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

PR knockout (PRKO) mice show higher bone mass Yao, PLoSONE, 2010 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 More dramatic HBM phenotype in females BV/TV

High bone mass in PRKO mice ? 1.Which cell population is responsible? 2.Which downstream signaling pathway of PR is responsible? 3.What is the mechanism of the sex differences? 4.The timing of PR knockout 1.PR regulates bone homeostasis within bone 2.The regulation has sex differences Hypothesis Further questions

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

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

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

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

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?

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

Bright field Green: GFP /Mx1 activity Red: Tomato /all others No induction Induced in vivo Bone marrow cells from the femurs, in vitro cultured for overnight Osteoclastic differentiation Blue: DAPI/nuclei Mx1-Cre; mTmG Col1-Cre; mTmG 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  stromal cells were differentiated into osteoblasts No differentiation 14d differentiation 1.Mx1-Cre is active in a part of osteoprogenitors before induction; 2.Mx1-Cre is harder to be activated in mineralized osteocytes? Mx1-Cre;mTmG mice, induced in vivo at 4 weeks of age

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

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