mesenchymal-to-epithelial
Recently,
it
was
reported that partial reprogramming of differentiated cells
using four reprogramming TFs (
Oct4, Sox2, Klf4
and
c-Myc
(OSKM))
in vivo
could generate tumors via epigenetic
Direct reprogramming can shed light on
cancer biology, and vice versa.
Transcriptional cascades definitively determine cell fate
during reprogramming to pluripotency and normal differentia-
tion. We examined whether murine cardiac mesenchymal
progenitors (CMPs)
expressing Sca-1 antigen and TFs
associated with cardiomyocytes can differentiate into adipo-
cytes and how the process is regulated. Elucidating the
mechanisms underlying adipose tissue generation in the heart
should help us to understand the pathophysiologies of
ischemic reperfusion injury (IRI) myocardial infarction (MI).
Results
Transduction of OSKM into CMPs is sufficient to induce
their differentiation into adipocytes.
To test our hypothesis
that reprogrammed CMPs can differentiate into other cell
types, we transduced CMPs with Sendai virus encoding
OSKM. We modified standard reprogramming medium by
removing leukemia inhibitory factor to avoid the generation
of iPSCs, following a previous report (Figure 1a).
Nine
days after infection (day 8), OSKM-transduced CMPs
(OSKM-CMPs) formed cytoplasmic lipid droplets, which were
not formed by untreated CMPs (CMP control) or CMPs
treated with adipogenic differentiation cocktails (CMP with
adipogenic cocktails) (Figure 1b). The lipid droplets in OSKM-
CMPs were clearly stained by Oil Red O (Figure 1b). Next,
to identify gene expression in reprogrammed CMPs, we
performed quantitative reverse transcription polymerase
chain reaction (qRT-PCR) analysis (Figure 1c). The expres-
sion levels of
Oct4
and
Sox2
in OSKM-CMPs decreased at
day 2 and were maintained at a low level thereafter.
Klf4
and
c-Myc
expression in OSKM-CMPs also decreased at day 2.
The expression levels of the adipogenic-related genes
C/Ebp
α
and
Fabp4
in OSKM-CMPs increased at day 4. The
expression levels of Fas, Ppar
γ
1 and Ppar
γ
2 in OSKM-CMPs
were higher than those in untreated CMPs at day 6.
Moreover, the expression levels of the cardiac-related genes
Mef2c
,
Gata4
and
Tbx5
in CMP controls increased, but these
genes were not expressed in OSKM-CMPs.
Transduction of OSKM into NIH3T3 fibroblasts is insuffi-
cient to induce their differentiation into adipocytes.
Next,
we transduced OSKM into NIH3T3 fibroblasts. At day 8,
NIH3T3 fibroblasts changed in shape from fibroblast-like cells
to round cells; however, there were no iPSC-like colonies or
Oil Red O-positive cells (Figure 2a). Expression of OSKM
genes at day 2 increased rapidly; however, the expression
levels of the adipogenic genes
Fas
,
C/Ebp
α
and
Ppar
γ
2
decreased steeply from day 2 (Figure 2b). In particular,
C/Ebp
α
and
Fas
expression did not differ from that of the
control (without OKSM). These results showed that OSKM-
transduced NIH3T3 fibroblasts did not differentiate into
adipocytes.
Microarray analysis of OSKM-CMPs.
To analyze global
gene expression in OSKM-CMPs, we performed microarray
analysis using an Agilent mouse microarray chip and the NIA
Array Analysis website.
Based on hierarchical clustering
analysis of gene expression, OSKM-CMPs could be clearly
discriminated from CMP controls (Figure 3a). In addition,
principal component analysis (PCA) of gene expression
showed that the OSKM-CMPs were different from the CMP
controls and gradually shifted from right to left on the PC1
axis in a time-dependent manner (Figure 3b). Furthermore, a
group of genes with decreasing expression over time
(positive direction along PC1, 4577 probes) and a group with
increasing expression over time (negative direction along
PC1, 5314 probes) were observed (Figure 3b). These genes
were categorized based on gene ontology (GO) annotations
and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathways (Figure 3c). Many genes showing decreasing
expression over time (PC1-positive direction) were assigned
to functional categories related to cell cycling and cell
division. In addition, many of the genes showing decreasing
expression over time were assigned to functions related to
focal adhesion and regulation of the actin cytoskeleton.
Otherwise, the genes showing increasing expression over
time (PC1-negative direction) were functionally related to
adipocyte differentiation, including saturated and unsaturated
fatty acid metabolism, fat cell differentiation and the PPAR
signaling pathway. These results strongly indicated that
OSKM-CMPs differentiated into adipocytes.
Klf4
and
c-Myc
have important roles in the differentiation
of CMPs into adipocytes.
To determine which of the
reprogramming factors among OSKM were critical for CMP
differentiation into adipocytes, we examined the effect of
removing each factor. We searched genetic databases for
information regarding gene expression during adipogenesis
in 3T3-L1 cells. The available information from previous
studies indicated that
Klf4
was expressed before adipogenic
stimulation and that
c-Myc
sharply rose up in response,
then the expression of both genes decreased to become
undetectable at 1 week, at which time lipid-laden adipocytes
were macroscopically recognized using the standard protocol
(GSE34150). Both
Klf4
and
c-Myc
have been reported to be
adipocyte differentiation-related factors.
We withdrew
OSKM sequentially. Neither the withdrawal of
Oct4
nor that
of
Sox2
influenced adipogenic differentiation based on Oil
Red O staining. The withdrawal of either
Klf4
or
c-Myc
decreased the percentage of Oil Red O-positive cells 9 days
after infection, compared with that observed when these
factors were present (Figure 4a). The expression levels of
genes related to adipogenesis including
C/Ebp
α
,
Fas
and
Ppar
γ
2
in TF(s)-transduced CMPs at day 8 showed a similar
pattern, regardless of the TFs used except OKM-infected
CMP. In OKM-infected CMP cells, the expression of both
C/Ebp
α
and Ppar
γ
1 was upregulated, but Fas, which is
involved in lipogenesis,
was downregulated. C/Ebp
α
is
mainly attributed to a role in insulin sensitivity in adipocyte
differentiation,
and Ppar
γ
2, but not Ppar
γ
1, has an essential
role in adipogenic differentiation
in vitro.
Overexpression
of c-MYC elicits p53-dependent apoptosis in primary
fibroblasts.
Infection of CMP cells might result in
Cardiac adiposity is regulated by
Klf4
and
c-Myc
D Kami
et al
2
Cell Death and Disease
