Download the role of mitogen activated protein kinase in transdifferentiation of

THE ROLE OF MITOGEN ACTIVATED PROTEIN KINASE IN TRANSDIFFERENTIATION OF ADIPOGENIC
MESENCHYMAL STEM CELLS
*Kim, E J; **Mok, J M; **Kim, Y H; *Kim, S J; **Hahn, S B; +**Lee, J W
*BK21 Project for Medical Sciences, Yonsei University. +**Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea.
Introduction
Bone marrow contains multipotential stromal stem cells that can
differentiate into fibroblastic, osteogenic, adipogenic and reticular cells.
These multipotential stem cells give rise to progenitors that display
apparent degree of plasticity or interconversion between the various cell
lines. However, the interrelationships between each of the fibroblastic,
osteogenic, adipogenic, and reticular lineages and the control mechanisms
governing the differentiation of each of the lineages remain unclear. The
decrease in bone volume in osteoporosis and age-related osteopenia is
accompanied with an increase in marrow adipose tissue. One mechanism
that could account for the apparent reciprocal relationship between
decreased bone density and increased fat formation is an imbalance in the
production of bone-forming and fat-forming cells in the marrow cavity.
Therefore, it is possible that inhibition of marrow adipogenesis with a
concomitant increase in osteogenesis could be a therapeutic target with
which to either prevent further increase in adipocyte formation or divert
existing adipocytes to become more osteoblatic with a resulting increase in
functional bone mass. Mitogen activated protein kinase (MAPK)dependent pathway is the signal transduction pathway that has the
potentiality in regulating the proliferation and differentiation of human
mesenchymal stem cells (MSCs). To characterize the molecular
mechanisms that regulate transdifferentiation of human MSCs, we examed
the contribution of MAPK family members, ERK, JNK and p38.
Materials and Methods
Cell culture: Human MSCs were harvested from bone marrow, and
isolated using Percoll gradient methods. Then MSCs were cultured in basal
medium (Dulbecco’s modified Eagle’s medium (DMEM)-low glucose
supplemented with 10 % fetal bovine serum (FBS) and 1 % antibioticantimycotic solution). Confluence was achieved within 7 days. Cells were
cultured in adipogenic medium (DMEM-low glucose supplemented with 1
% antibiotic-antimycotic solution plus 0.5 mM isobutyl-methylxanthin, 1
µM dexamethasone, 10 µM insulin, 200 µM indomethacin) in the presence
of 25 µM troglitazone for 14 days until over 80 % of the cells
differentiated adipogenesis. Adipogenic medium was changed osteogenic
medium (DMEM - high glucose supplemented with 10 % FBS and 1 %
antibiotic-antimycotic solution plus 100 mM dexamethasone, 10 mM βglycerophosphate, and 50 µg/ml ascorbic acid-2-phosphate) and cultured
for 3, 7, 14 and 21 days.
Stain: Using the Oil-red-O, von kossa and alkaline phosphatase (ALP)
solutions, stains were done.
Semi-quantitative reverse-transcription polymerase chain reactions (RTPCR): RT-PCR was performed to detect RNA expression of lipoprotein
lipase (LPL), fatty acids binding protein (aP2), peroxisome proliferatoractivated receptor-γ (PPARγ), collagen type I and osteocalcin. Total RNA
was isolated from cell pellets by the RNeasy kit (Qiagen, Hilden,
Germany). Total RNA was reverse-transcribed with use of the Omniscript
kit (Qiagen). Aliquots of the resulting cDNA were amplified in a total
volume of 50 µhopl containing polymerase chain reaction cocktail
(Qiagen). Integrated density values for the genes were then normalized to
GAPDH values.
Western blot analysis: To characterize the molecular mechanisms that
regulate transdifferentiation, we examined the contribution of MAPK
family members, ERK, JNK, and p38 by Western blot analysis. Cell
lysates were clarified by centrifugation and the protein content of
supernatants was determined using the modified Bradford assay. Equal
amount (30 µg) of protein per sample was load onto a 4 % stacking gel and
6 % polyacrylamide mini-gel and electrophoreses in SDS running buffer.
Then, Proteins were electrophoretically transferred onto a Hybond-P
membrane (Armersham, Buckinhamshire, UK) for overnight. Membranes
were blocked for 1 h and primary antibodies (ERK, JNK, p38, P-ERK, PJNK, and P-p38) (Santa Cruz, Santa Cruz, CA) were incubated with the
membranes for 3 hours. After washing, membranes were incubated for 1
hour with appropriate horseradish peroxidase-conjugated secondary
antibody (Santa Cruz) and signals were visualized using ECL detection
reagents ( Armersham).
Results
In RT-PCR, when adipogenic MSCs were cultured in osteogenic medium,
mRNA levels for the adipocyte gene markers (LPL, aP2, and PPARγ) were
decreased and mRNA levels for the osteoblast gene markers (collagen type
I and osteocalcin) were increased (Fig 1). Large numbers of adipocytes in
osteogenic medium for 21 days exhibited a fibroblastic appearance (Fig 2)
and stained positively with von kossa and ALP in time-dependent manner
(data not shown). In Western blot analysis, we founded that osteoinductive
culture medium strongly activated ERK, JNK, and p38 in a time-dependent
manner (data not shown).
Fig 1. The total RNA harvested from human MSC cultures in the presence
of adipogenic medium was examined through semi-quantitative RT-PCR.
Fig 2. Morphology of differentiated and undifferentiated human MSCs.
Adipogenic MSC transdifferentiation observed during culture in
osteogenic medium (Original magnification X200).
Discussion
In the present study, adipogenic MSCs were shown to differentiate to the
osteogenic phenotypes under appropriate culture conditions. Based on our
findings of the inverse regulation of osteogenesis and adipogenesis by
MAPK in human MSCs, we suggest a role for ERK, JNK and p38 as a
regulatory switch for these differentiation pathways. Furthermore,
evidence of the transdifferentiation of these cells suggests that a large
degree of plasticity exists between osteoblasts and adipocytes and,
significantly, that they have reciprocal relationship between adipogenesis
and osteogenesis. In order to obtain more precise information about the
plasticity of the osteogenic and adipogenic phenotype, pure adipocytes
have been prepared from human bone marrow.
Acknowledgement
This study was supported by the BK21 Project for Medical Sciences,
Yonsei University and by a special grant of the Dean of Yonsei University
College of Medicine. (2001-01)
49th Annual Meeting of the Orthopaedic Research Society
Poster #0876