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 orAdrenal glands

Progesterone

?

Sex hormones for bone homeostasis

osteoblastosteoclast

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+Col2α1+

Col2α1+

Runx2+ColXα1+

Chondrocyte Hypertrophicchondrocyte

Runx2+

Osteoblast progenitor

Osteoblast precursor

Osx1+

Osteoblast precursor

Oc+

Osteoblast

CapG+Dmp1+

Mineralizing osteocyte

SOST+FGF23+ORP150

Matureosteocyte

Figure adapted from Bonewald,2011, Vankoevering, 2008 and Rodda, S. J. 2006

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-CreOsx1-Cre Osteoclast

TRAP-CreCtsK-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

XPR-flox/flox

Tg/+, flox/flox

Tg/+, flox/+

1. Mx1-Cre Osteoprogenitors 2. Col1a1-CreERT2Mature osteoblasts

PR is knocked out in “osteoprogenitors” or “mature osteoblasts”

Induce

pI-pC orTamoxifen

XMore tg/+, flox/flox +/+, flox/flox

Mx1-Cre;PR-flox heterozygotes

Tg, flox/+, FemalepI-pC IP injection starts at 1 month of age

TV BV TV/BVwt 2.70 0.20 0.08

PRKO 2.41 0.61 0.25

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

loxPEGFP

loxPtdTomato

Stop codon 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 fieldGreen: GFP /Mx1 activityRed: Tomato /all others

No induction

Induced in vivo

Bone marrow cells from the femurs, in vitro cultured for overnight

Osteoclastic differentiationBlue: 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