1. Validation of osteocyte-specific growth hormone
receptor (GHR) null mouse model
Xiangyu Peng, Hui Sun, YingJie Wu, and Shoshana Yakar
New York University COLLEGE OF DENTISTRY , David B. Kriser Dental Center , Department of Basic Science
and Craniofacial Biology
INTRODUCTION
Osteocytes, the most abundant cells in bone, orchestrate skeletal growth andOsteocytes, the most abundant cells in bone, orchestrate skeletal growth and
remodeling via secretion of endocrine and autocrine/paracrine modulators andremodeling via secretion of endocrine and autocrine/paracrine modulators and
cross talk with osteoblasts and osteoclasts on bone surfaces. Osteocytes arecross talk with osteoblasts and osteoclasts on bone surfaces. Osteocytes are
versatile cells, which interact with each other via dendritic processes and canversatile cells, which interact with each other via dendritic processes and can
remodel their perilacunar matrices in response to physiological demands.remodel their perilacunar matrices in response to physiological demands.
Growth hormone (GH) and the insulin-ike growth factor-1 (IGF-1) play essentialGrowth hormone (GH) and the insulin-ike growth factor-1 (IGF-1) play essential
roles in skeletal acquisition. In a series of studies we have demonstrated thatroles in skeletal acquisition. In a series of studies we have demonstrated that
serum levels of IGF-1 play important roles in transversal bone growth, andserum levels of IGF-1 play important roles in transversal bone growth, and
postulated that GH/IGF-1 also play roles in osteocytes function during growth.postulated that GH/IGF-1 also play roles in osteocytes function during growth.
The IGF-1 receptor, insulin receptor and GH receptor (GHR) have not beenThe IGF-1 receptor, insulin receptor and GH receptor (GHR) have not been
reported in connection with osteocytes during skeletal development.reported in connection with osteocytes during skeletal development.
Nonetheless, in response to mechanical loading or fracture, a marked increaseNonetheless, in response to mechanical loading or fracture, a marked increase
in osteocytic IGF-1 mRNA levels can be detected.in osteocytic IGF-1 mRNA levels can be detected.
To better understand how bone intrinsic GH/IGF-1 signaling regulate skeletalTo better understand how bone intrinsic GH/IGF-1 signaling regulate skeletal
acquisition, geometric structure and strength, we have generated osteocyte-acquisition, geometric structure and strength, we have generated osteocyte-
specific GHR inactivation using the Cre/loxP system, where the Crespecific GHR inactivation using the Cre/loxP system, where the Cre
recombinase was driven by the dentin matrix protein (DMP)-1 promoter (10Kb).recombinase was driven by the dentin matrix protein (DMP)-1 promoter (10Kb).
METHODS
DNA extraction
DNA was extracted from different tissues. The samples were digested in lysis
buffer with 20mg/ml proteinase K (100mM Tris pH=8.0, 200mM NaCl, 5mM
EDTA pH=8.0, 0.20% SDS). Consequently, DNA was precipitated with
isopropanol, washed in 70% ethanol, and dissolved in water. PCR was
performed using the R2 and S6 primers with annealing temperature of 57℃.
RNA extraction and real-time PCR
RNA was extracted from bone and muscle using QIAzol reagent and reverse
transcribed according to the manufacturer’s instructions (Qiagen, Valencia,
CA; Invitorgen Corp., Carlsbad, CA). Real time-PCR was performed using the
QuantitectTM
SYBR®
green PCR kit (Qiagen, Valencia, CA) in the ABI PRISM
7900HT sequence detection system (Applied Biosystems, Foster City, CA).
Each transcript in each sample was assayed 3 times and the fold-change
ratios between experimental and control samples were calculated relative to
b-actin, GAPDH or 18S.
Protein extraction
Mice were injected intraperitoneally with 5mg/kg GH and tissues harvested 15
min later. Protein was extracted in the following lysis buffer: 50Mm Tris, pH
7.4; 150mM NaCl; 1mM EDTA); 1.25% CHAPS; 20mM NaF; 10mM Na
pyrophosphate; 8mM β-glycerophosphate; 1mM Na orthovanadate;
Complete® Protease Inhibitor Cocktail [Roche]. Then 25-30ug of protein were
separated on 4-20% acrylamid gel and transferred to a nitrocellulose
membrane. Western immunoblot assay was used to detect total and
phosphorylated STAT5 proteins. The primary antibodies used were anti-
phospho-Stat5Tyr694
(pSTAT5), anti-STAT5, anti b-actin, and HRP-linked anti-
rabbit antibodies (Cell Signaling Technology, Danvers, MA).
RESULTS
CONCLUSIONS ACKNOWLEDGMENT
S:
GOAL
Our initial goal was to validate whether DMP-derived CreOur initial goal was to validate whether DMP-derived Cre
mediates osteocyte-specific growth hormone receptormediates osteocyte-specific growth hormone receptor
knockout (GHRKO) specifically in osteocytes.knockout (GHRKO) specifically in osteocytes.
The DMP-GHRKO is a valid mouse model to study the role of GHR specifically in osteocytes. The ghr
gene recombination was not restricted to bone, but was found in muscle as well. Thus, when
characterizing the bone phenotype of this model we should note that gene recombination was detected
also in muscle.