1. PP5, Brokering Energy and
Architecture
Ali Eltatawy
Doctor of Medicine Candidate
Dr. Beata Lecka-Czernick, Phd.
Department of Orthopedic

2. Introduction
Mesenchymal stem cells (MSC) arrive at a fork in the road where one path leads to fat
and energy metabolism and the other path leads to bone formation and architecture.
PPARγ and RUNX2 are reciprocal regulators of MSC differentiation toward adipocytes
and osteoblasts, respectively.

3. Ongoing studies in the lab have determined that Protein phosphatase5 (PP5) possesses
uniquebiochemical properties which regulate PPARγ and RUNX2 phosphorylationsites
and is implicated as the broker between reciprocal processes of fat and boneformation.

4. Mice deficient in PP5 have
shown overall increased
osteoblast numbers, high bone
formation, and a decrease in lipid
accumulating adipocytes in the
bone marrow.

5. Rosiglitazone (Rosi) is a drug in the
thiazolidinedione (TZD) class for
treatment of Type 2 diabetes mellitus
(T2DM), which binds and activates
PPARγ nuclear receptor in fat cells;
leading to trabecular bone loss,
increased risk of fractures and fat
deposition in bone marrow

6. Ongoing studies show that mice deficient in PP5 may be protected from the negative
effects of Rosi.
It is not known how PP5 activity regulates the balance between energy
and bone architecture and how its roles differ in tissues throughout the
body. Experiments aim to phenotypically characterize mice with a
tissue specific PP5 gene knockout and better understand the PP5 role in
bone and adipose tissue.

7. Methods
The initial goal is to genetically profile mice through mouse-tail genotyping. This
protocol includes DNA isolation, polymerase chain reaction followed by gel
electrophoresis. The lab profiled 4 kinds of control mice including Adipo Cre, PRX Cre,
PP5 Flox/Floxand wildtype. The 2 strains of mice that have tissue specific PP5 knockout
are AdipoCre PP5 Flox/Flox(A.Cre fl/fl) and PRX Cre PP5 Flox/Flox(P.Cre fl/fl). The
A.Cre fl/fl results in loss of PP5 in adipose whereas P.Cre fl/fl results in loss of PP5
specifically in long bones. The animal treatment and care protocols conformed to NIH
Guidelines and were performed using a University of Toledo Health Science Campus
Institutional Animal Care and Utilization Committee protocol.

8. Mouse dissectionwas used to extract blood, white adipose tissue (WAT), brown adipose
tissue (BAT), liver, tibia midshaft, femur and spine for further analysis. Serum was
isolated from the blood.
BAT
Liver
WAT
Bone

9. The bone marrow was extracted from the tibia then osteoblast and osteocyte fractions
were separated for further analysis.
Bone cleaningandCT prep Bone marrow spin down extraction
CT ready

10. Microcomputed tomography (mCT) of the tibiae and L4 vertebrae was performed
using the μCT-35 system (Scanco Medical AG, Bassersdorf, Switzerland). Briefly, scans
were performed at 70 kV, energy and 113 µA intensity and using 7 µm voxel. Images of
trabecular bone were segmented at 289 threshold value using per mille scale. The
analysis of bone microstructure conformed to recommended guidelines.
Sample Scanning Contouring
&
Cleaningspine andtibia
Mouse foot before
cleaning
Tibia CT
Vertebrae CT
Trabecular CT

11. Results
A.Cre fl/fl mice had lower overall weights, increased BAT weight and no change in
WAT or liver weight. P.Cre fl/fl mice had higher overall weight, higher WAT and no
change in BAT or liver weight.
mCT analysis of proximal tibia (long bones) A.Cre fl/fl showed an increased trabecular
number (TbN) and Bone Volume/ Trabecular Volume (BV/TV), decrease in trabecular
spacing (TbSp), but no change to trabecular thickness (TbTh). P.Cre fl/fl mice proximal
tibia showed an increase in both TbTh and BV/TV but no change in TbN or TbSp.

12. mCT analysis of vertebrae (non-long bone) A.Cre fl/fl showed an increased TbN and
BV/TV, a decreased TbSp, but no change to TbTh. However, P.Cre fl/fl mice vertebrae
showed no change throughout.
mCT analysis of tibia midshaft (cortical bone) P.Cre fl/fl showed an increase in cortical
thickness and bone mineral density. However, A.Cre fl/fl mice tibia midshaft showed no
change throughout.

13. Conclusion
The P.Cre fl/fl mice show an increase in trabecularthickness in long bones and a thicker
cortical shaft which implicates PP5 in bone as a direct effector on MSC lineage
commitment. The A.Cre fl/fl mice show an increased trabecularnumberand decrease
trabecularspace demonstrating adipose PP5’s anaboliceffect on the bone. PP5 is
regulating the differentiation of MSCs to adipocytes and osteoblastthrough PPARγ and
RUNX2 phosphorylationand is also a regulator of adipocytes and osteoblasticactivity.