1: Stem Cells. 2005 Sep;23(8):1113-21. Epub 2005 Jun 13. 

Temporal expression patterns and corresponding protein inductions of early
responsive genes in rabbit bone marrow-derived mesenchymal stem cells under
cyclic compressive loading.

Huang CY, Reuben PM, Cheung HS.

Research Service, Miami VA Medical Center, 1201 NW 16th Street, Miami, Florida
33125, USA.

Our recent study suggested that cyclic compressive loading may promote
chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose
cultures through the transforming growth factor (TGF)-beta signaling pathway. It
has been shown that the activating protein 1 (AP-1) (Jun-Fos) complex mediated
autoinduction of TGF-beta1 and its binding activity was essential for promoting
chondrogenesis of mesenchymal cells, whereas Sox9 was identified as an essential
transcription factor for chondrogenesis of embryonic mesenchymal cells. The
objective of this study was to examine temporal expression patterns of early
responsive genes (Sox9, c-Fos, c-Jun, and TGF-beta type I and II receptors) and
induction of their corresponding proteins in agarose culture of rabbit BM-MSCs
subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from
the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were
made by suspending BM-MSCs in 2% agarose gel (10(7) cells/ml) for cyclic,
unconfined compression tests performed in a custom-made bioreactor. In the
loading experiment, specimens were subjected to sinusoidal loading with a
magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day.
Experiments were conducted for 2 consecutive days. This study showed that cyclic
compressive loading promoted gene expressions of Sox9, c-Jun, and both TGF-beta
receptors and productions of their corresponding proteins, whereas those gene
expressions exhibited different temporal expression patterns among genes and
between 2 days of testing. The gene expression of c-Fos was detected only in the
samples subjected to1-hour dynamic compressive loading. These findings suggest
that the TGF-beta signal transduction and activities of AP-1 and Sox9 are
involved in the early stage of BM-MSC chondrogenesis promoted by dynamic
compressive loading.

PMID: 15955834 [PubMed - in process]


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2: J Cell Biochem. 2003 Apr 15;88(6):1129-44. 

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged
and tight regulation of ERK1/2.

Seghatoleslami MR, Roman-Blas JA, Rainville AM, Modaressi R, Danielson KG, Tuan
RS.

Division of Rheumatology, Department of Medicine, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107, USA. reza.seghatoleslami@mexil.tju.edu

Close contact of mesenchymal cells in vivo and also in super dense micromass
cultures in vitro results in cellular condensation and alteration of existing
cellular signaling required for initiation and progression of chondrogenesis. To
investigate chondrogenesis related changes in the activity of ubiquitous cell
signaling mediated by mitogen-activated protein kinases (MAP kinase), we have
compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells
as monolayers (non-chondrogenic culture) or high density micromass cultures
(chondrogenic) on the regulation and phosphorylation state of extracellular
signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2
nuclear targets, namely, activation protein-1 (AP-1) and serum response factor
(SRF). Increasing cell density resulted in reduced DNA binding as well as
activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent
monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels
of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed
induction of AP-1 and SRF activity whereas higher concentrations of this
inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations
of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day)
resulted in differential regulation of c-Fos and c-Jun protein levels as well as
total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of
C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen
and Sox9 gene expression. While high expression of aggrecan and type IIB
collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2
treated micromass cultures resulted in reduced activity of both genes. Our
findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2
cells is tightly controlled and can cross interact with other signaling
activities mediated by BMP-2 to positively regulate chondrogensis.

PMID: 12647296 [PubMed - indexed for MEDLINE]


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