Mol Cell Biochem (2018) 437:133–142
https://doi.org/10.1007/s11010-017-3101-2
Cytoglobin inhibits migration through PI3K/AKT/mTOR
pathway in fibroblast cells
Selami Demirci1,2 • Ayşegül Doğan1,3 • Hüseyin Apdik1 • Emre Can Tuysuz1,4
Sukru Gulluoglu1,4 • Omer Faruk Bayrak4 • Fikrettin Şahin1
Received: 12 April 2017 / Accepted: 9 June 2017 / Published online: 15 June 2017
Ó Springer Science+Business Media, LLC 2017
Abstract Cell proliferation and migration are crucial in
many physiological processes including development,
cancer, tissue repair, and wound healing. Cell migration is
regulated by several signaling molecules. Identification of
genes related to cell migration is required to understand
molecular mechanism of non-healing chronic wounds
which is a major concern in clinics. In the current study, the
role of cytoglobin (CYGB) gene in fıbroblast cell migra-
tion and proliferation was described. L929 mouse fibroblast
cells were transduced with lentiviral particles for CYGB
and GFP, and analyzed for cell proliferation and migration
ability. Fibroblast cells overexpressing CYGB displayed
decreased cell proliferation, colony formation capacity, and
cell migration. Phosphorylation levels of mTOR and two
downstream effectors S6 and 4E-BP1 which take part in
PI3K/AKT/mTOR signaling declined in CYGB-overex-
pressing cells. Microarray analysis indicated that CYGB
overexpression leads to downregulation of cell prolifera-
tion, migration, and tumor growth associated genes in L929
cell line. This study demonstrated the role of CYGB in
fibroblast cell motility and proliferation. CYGB could be a
& Ayşegül Doğan
aysegul.dogan@nih.gov; aguldgn@gmail.com
1
Department of Genetics and Bioengineering, Faculty of
Engineering and Architecture, Yeditepe University,
Kayisdagi Cad. 26 Agustos Yerlesimi,
34755 Atasehir, Istanbul, Turkey
2
National Heart, Lung, and Blood Institute (NHLBI), NIH,
Bethesda, MD, USA
3
National Cancer Instıtute, CDBL, NIH, Frederıck, MD, USA
4 steps in wound healing among several events including
Department of Medical Genetics, Yeditepe University
Medical School Inonu Mah, Kayisdagi Cad. 26 Agustos
Yerlesimi, 34755 Atasehir, Istanbul, Turkey
•
promising candidate for further studies as a potential target
for diseases related to cell migration such as cancer and
chronic wound treatment.
Keywords Cytoglobin  Cell migration  Wound healing 
Microarray  Tumor suppressor
Introduction
Cytoglobin (CYGB) was originally characterized in 2001 as
the fourth globin in mammals in molecular analysis of acti-
vated rat hepatic stellate cells (HSCs); hence, it was termed as
stellate cell activation-associated protein (STAP) [1, 2]. After
its discovery, the scientific community has paid considerable
attention to CYGB as it has been associated with oxygen
storage, transport, and sensing along with reactive oxygen
storage elimination [3]. In addition, loss of function for
CYGB has been linked to proliferation and migration abilities
of cancer cells and organ fibrosis, confirmed by CYGB
knockout mice and cell lines [4, 5], proposing that CYGB
could be referred as tumor suppressor gene. Unlike other
globin family proteins, myoglobin, hemoglobin, and neu-
roglobin, CYGB has been detected in various organs
including liver, heart, brain, lung, retina and gut, and in
several organ-specific cell types such as neurons, macro-
phages, muscles, hepatocytes, epithelial cells, chondroblasts,
osteoblasts, and fibroblasts [6]. Although its localization in
epithelium and fibroblastic tissue has been revealed, the role
of CYGB on wound healing, especially in non-healing
chronic wounds, has not been investigated so far.
Fibroblast proliferation and migration are the critical
inflammation, angiogenesis, cell migration, contraction, and
tissue remodeling. Besides, fibroblast migration is crucial to
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all of these events as it provides the collagen tissue at the
wound region [7]. Growth factor-induced fibroblast acti-
vation leads cell movement to the wound area and synthesis
of collagen-rich matrix. Then, a-smooth muscle actin (a-
SMA) expressing fibroblasts differentiate into myofibrob-
lasts providing contractile forces for an effective wound
closure [8]. Migration of fibroblasts is regulated by several
growth factors and cytokines including platelet-derived
growth factor-AA (PDGF-AA), transforming growth factor
(TGF)-b1, fibroblast growth factors FGFs, and interleukins
(ILs) [9]. Understanding the possible molecular mechanism
of cell migration and regulatory genes at the molecular level
might lead to the identification of new therapeutic modali-
ties for efficient therapies, especially for cancer or non-
healing chronic wounds. Cutaneous wound healing is a
dynamic and complex process in which several growth
factors, cytokines, extracellular matrix, and skin cells
including fibroblasts, keratinocytes, epithelial and
endothelial cells are involved in a tightly controlled manner
[8, 10]. Fibroblast cell migration coupled with proliferation
is required for the repair of injured skin and wound healing.
Fibroblast cells play an important role in the wound-healing
process by regulating molecular pathways that maintain
growth factor secretion and tissue dynamics through cell
proliferation and motility. Understanding of the molecular
mechanisms that control the cell migration and proliferation
of fibroblasts cells is necessary to develop new therapies to
manage tissue regeneration and complete wound healing.
Activated PI3K pathway and downstream effectors have
multifactorial and cooperative effects on cell migration,
extracellular matrix maturation, and wound healing [11].
PI3K-Akt-mTOR pathway is not only involved in cell
proliferation and migration, but also regulates the protein
synthesis in the cells to control metabolism [12]. Although
aberrant activation of the PI3K-Akt-mTOR pathway is
generally observed in malignancies, this pathway acts as a
component of wound-healing process [13].
In the current study, proliferation and migration kinetics
of CYGB-overexpressing L929 mouse fibroblast cells were
evaluated using in vitro approaches. CYGB was assessed
for its modulating activity on the fibroblast cell behavior
in vitro by decreasing their migratory and proliferation
potential in a mechanism that involves the downregulation
of the PI3K/Akt/mTOR signaling.
Materials and method
Preparation of lentiviral gene constructs
The coding sequence of mouse CYGB (2281-bp) was lib-
erated by digestion with NotI and EcoRV and ligated into
pLenti-III-2A-GFP (Abm, Richmond, CA, USA, Fig. 1a).
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Mol Cell Biochem (2018) 437:133–142
Lentiviral vector stocks for GFP and CYGB-expressing
vectors were prepared by calcium phosphate transfection of
293T cells with pLenti-III-CYGB-2A-GFP or pLenti-III-
2A-GFP, pCMVDR8.2DVPR (Addgene, Cambridge, MA,
USA), and pMD2.G (VSVG, Addgene, Cambridge, MA,
USA). Viral supernatants were harvested at 48 and 72 h
post transfection, filtered through 0.45-lm filter, and con-
centrated by ultracentrifugation. The titers were calculated
by infection of Hela cells and analysis of GFP-positive
cells by flow cytometry 72 h after infection. The titers of
GFP- and CYGB-expressing vector stocks were 4 9 104
transducing units/ml.
Cell culture and transduction
L929 mouse fibroblast cells (NCTC clone 929 ATCC
CCL-1) were maintained Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen, Carlsbad, CA) supple-
mented with 10% FBS and 1% PSA at 37 °C and 5% CO2
in a humidified incubator. Post-transfection of L929 cell
with viral supernatants of GFP and CYGB was performed
in the presence of 8 lg/ml of polybrene and fresh media
at 1:1 ratio for 24 h. Transduced cells were selected with
2 lg/ml puromycin treatment and GFP-positive cells
were sorted by flow cytometry (FACS Aria, Becton–
Dickinson, San Jose, CA). CYGB overexpression in
transduced cells was confirmed by RT-PCR and western
blot analysis.
Cell viability, proliferation, and doubling assays
Cell viability of L929-GFP and L929-CYGB was measured
by the 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxy-meth-
oxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)
assay (CellTiter96 Aqueous One Solution; Promega,
Southampton, UK) as described previously [14]. Briefly,
5 9 103 cells were seeded onto 96-well plates and cell
viability was analyzed for 24, 48, and 72 h. At the end of
each time interval, cells were incubated with 10 ll of MTS
reagent and 100 ll of growth medium for 2 h at 37 °C.
Absorbance at 490 nm was detected using an ELISA plate
reader (Biotek, Winooski, VT).
Cell proliferation was determined as described previ-
ously [15]. L929-GFP and L929-CYGB cells were seeded
onto 12-well plates in triplicate at a density of 1 9 104
cells per well and medium was replaced every other day.
Cells were stained with trypan blue and cell number was
counted using a hemocytometer. Cell doubling was cal-
culated according to the modified protocol as described
previously [16]. L929-GFP and L929-CYGB cells were
seeded onto 12-well plates at a cell density of 1 9 104
cells per well, counted every day to determine doubling
time.
Mol Cell Biochem (2018) 437:133–142 135

Fig. 1 Effect of CYGB

overexpression on cell
proliferation, viability, and
colony formation. a Plasmid
construct used in
overexpression studies,
b confirmation of CYGB
overexpression by RT-PCR, and
c western blot. d Cell counts of
L929-GFP and L929-CYGB for
day 1, day 2, and day 3. CYGB
overexpression blocks cellular
proliferation. e Cell doubling
time for L929-GFP and L929-
CYGB cells. CYGB
overexpression increased
doubling time for fibroblast
cells compared to GFP-
expressing control group. f Cell
viability analysis by MTS for
day 1, day 2, and day 3 for
L929-GFP and L929-CYGB
cells. Cell viability was
significantly decreased in L929-
CYGB at day 2 and day 3.
g Colony-forming capacity of
L929-GFP and L929-CYGB.
Colonies were stained with
crystal violet and counted. The
number and diameter of
colonies were considerably low
in L929-CYGB cells. Notes
* p \ 0.05

Colony-forming unit (CFU) assay
CFU assay was performed to analyze whether CYGB over-
expression changes the colony-forming ability of cells [17].
Briefly, cells were diluted in culture medium and seeded at a
cell density of 80 cell per well of 6-well plates and incubated
for 14 days at 37 °C in a humidified incubator. Colonies were
stained with crystal violet and counted.
Cell migration analysis
In order to evaluate the effect of CYGB overexpression in
fibroblast cells, in vitro scratch and trans-well cell migration
assays were performed. L929-GFP and L929-CYGB cells
were seeded onto a 12-well plate (TPP, Switzerland) at a cell
density of 1 9 105 cells/well and incubated overnight in a
humidified incubator at 37 °C and 5% CO2. Then, cells were
scratched using a sterile 200-ll pipet tip and washed with
PBS, and cell culture medium was immediately replaced
with fresh medium. Cell migration to the wounded area was
determined by pictures taken after 24 h using Zeiss Pri-
moVert light microscope with an AxioCam ERc5 s camera
and Zen 2011 software (Carl Zeiss Microscopy, LLC,
Thornwood, NY, USA) and migrated cell numbers to the
wounded area was measured using image analysis software
(ImageJ, NIH, Bethesda, MD). The scratch area was

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measured at different time points (0 h and 24 h) and
% scratch closure was calculated by the formula:
Scratch area at 0 h  Scratch area at 24 h
Scratch closureð%Þ ¼
Scratch area at 0 h
 100:
Secondly, trans-well cell migration analysis was con-
ducted using CytoSelectTM 24-well kit (CBA-100-C, Cell
Biolabs Inc., San Diego, CA, USA) according to the manu-
facturer’s instructions. In short, L929-GFP and L929-CYGB
cells were seeded onto trans-wells with 8 lm pores (upper
chamber) at a cell density of 3 9 104 cells/well in serum-free
DMEM. DMEM supplemented with 10% FBS was used as
chemoattractant media to trigger cell migration. After 24 h
incubation period, cells were stained with cell stain solution
provided by kit and pictures were taken by light microscope.
Migration was quantitatively analyzed by dissolving the dye
in the extraction solution supplied by the kit. Briefly, the dye
was dissolved by adding 200 ll of extraction solution to each
well and incubating 10 min. Absorbance of the samples were
taken at 560 nm in a plate reader and analyzed.
Western blot analysis
Primary antibodies against AKT/p-AKT (#9272), S6/p-S6,
mTOR/p-mTOR (#5536), PTEN (#9559), p-4E-BP1 (#2855),
GAPDH (#8884), purchased from Cell signaling technology
(Beverly, MA, USA), p53 (sc-6243-Santa Cruz), p21 (sc-397-
Santa Cruz), and primary antibodies against pro-caspase3/
active caspase (04-440-Millipore) were used to detect marker
proteins for apoptosis and AKT pathway in L929-GFP and
L929-CYGB cells. Briefly, protein samples were loaded to
Any kDTM Mini-PROTEANÒ TGXTM precast gels (#456-
9033, Bio-Rad, USA) at a concentration of 30 lg/lane and
transferred to nitrocellulose membranes (#162-0115, Bio-
Rad, Germany). Membranes were incubated with primary
antibodies (dilution 1:5000) at 4 °C for 16 h. Then, mem-
branes were incubated with respective anti-rabbit or anti-
mouse secondary antibody (sc-2004, sc-2002 dilution
1:10,000, Santa Cruz Biotech Inc., USA) prepared in blocking
buffer for 1 h at room temperature. GAPDH was used as an
internal control and images were taken using the luminometer
system (Bio-Rad, USA). Band intensities were calculated
using Image J software and normalized to the respective
GAPDH band intensities. Results were represented as fold
change of control.
RT-PCR analysis
Quantitative RT-PCR experiments were performed accord-
ing to the previously described protocol [18]. Primers for
CYGB and GAPDH were designed using Primer-BLAST
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Mol Cell Biochem (2018) 437:133–142
software from the National Center for Biotechnology
(Bethesda, MD, USA) and synthesized by Macrogen (Seoul,
Korea). GAPDH was used as housekeeping gene. Total RNA
from L929-GFP and L929-CYGB cells were isolated using
RNAeasy plus mini kit (Qiagen, Hilden, Germany) and
cDNA was synthesized using High Fidelity cDNA synthesis
kit (Roche, USA). Reverse transcription polymerase chain
reaction (RT-PCR) with SYBR Green method was used to
detect mRNA levels of the target genes. All RT-PCR
experiments were conducted using CFX96 RT-PCR system
(Bio-Rad, Hercules, CA).
Microarray analysis
Total RNA from L929-GFP and L929-CYGB cells at 80%
confluency were isolated using TRIzol Reagent (Invitrogen,
USA) and transcriptome analysis was conducted with Mouse
Gene 2.1 ST Array (Affymetrix, GeneAtlas system)
according to the manufacturer’s protocol. Resulting data
were analyzed by Transcriptome analysis console 3 software
(Affymetrix). Differentially expressed genes were selected
based on ± 1.5-fold difference and p value of less than 0.05.
Statistical analysis
The data were statistically analyzed using one-way analysis
of variance and Tukey post hoc test. The values of
p \ 0.05 were considered statistically significant.
Results
CYGB overexpression inhibits cell proliferation, cell
viability, and CFU formation
CYGB overexpression was observed in CYGB-L929 cells in
comparison with GFP transduced cells by gene and protein
expression analysis (Fig. 1b, c). L929-GFP cells were grad-
ually proliferating and increasing in cell number, whereas cell
proliferation rate of L929-CYGB cells was relatively lower
(Fig. 1d). While the initial 1 9 105 cell number was increased
to 1.3 9 106 for L929-GFP cells, 4.5 9 105 cells for L929-
CYGB group were counted at the end of 4 days of propagation
period. This observation was confirmed by cell doubling
assay. Doubling time was 19 h and 33 h for L929-GFP and
L929-CYGB, respectively (Fig. 1e).
Cell viability of L929-GFP and L929-CYGB was mea-
sured by MTS assay for 24, 48, and 72 h. Cell viability of
L929-CYGB was significantly reduced at day 2 and day 3
(Fig. 1f). Countable colony number was scarce in L929-
CYGB cells compared to L929-GFP (Fig. 1g). Although
L929-CYGB cells had 17 countable colonies, L929-GFP
cells formed 82 colonies quantitatively.
Mol Cell Biochem (2018) 437:133–142
CYGB overexpression inhibits cell migration
in vitro
Fibroblast cells were subjected to migration analysis in
order to observe migration ability in transduced cells.
Scratch assay analysis showed that CYGB overexpression
suppressed the cell migration to the wounded area
(Fig. 2a). There was almost a 2-fold decrease at scratch
closure in CYGB transduced cells measured by quantita-
tive analysis (Fig. 2b). Similar results were observed in
trans-well membrane migration analysis. Although most of
the L929-GFP cells transferred through the membrane in
24 h, L929-CYGB cells were failed to migrate from trans-
wells (Fig. 2c). It was noted that 2-fold decrease in the
migration of CYGB transduced cells was observed by OD
measurements compared to GFP-positive cells (Fig. 2d).
CYGB blocks AKT pathway and triggers apoptotic
pathway in L929 fibroblast cells
As CYGB prevented cell proliferation and migration,
blockage of cell survival pathways including AKT-
Fig. 2 Effect of CYGB overexpression on fibroblast cell migration
analyzed by scratch and trans-well cell migration assays. a Micro-
scopic evaluation of cell migration in vitro scratch assay. b Quanti-
tative measurement of scratch closure. CYGB overexpression inhibits
cellular motility and decreased scratch closure rate. c Cell migration
137
mediated pathway and activation of apoptosis was
hypothesized in CYGB-L929 cells. Therefore, we exam-
ined the expression levels of total and phosphorylated AKT
as this signaling is activated by PI(3)K and inhibited by
PTEN. Forced expression of CYGB regulated downstream
pathway by triggering the PTEN expression. Overexpres-
sion of CYGB reduced the phosphorylation of AKT which
might be through induction of PTEN expression (Fig. 3a).
Since the role of mTOR in cytoskeleton regulation [19] has
been shown, we next demonstrated whether CYGB over-
expression inhibits migratory characteristics of cells by
affecting the mTOR phosphorylation and two downstream
effectors S6 and 4E-BP1. As expected, phosphorylation of
mTOR, S6, and 4E-BP1 reduced in L929-CYGB indicating
that CYGB overexpression decreases cell migration
through suppression of mTOR kinase activity and resulting
diminished S6 and 4E-BP1 signaling (Fig. 3a).
Tumor suppressors p53 and p21 were upregulated in
L929-CYGB cells which indicates the activation of apop-
tosis and inhibition of cell proliferation (Fig. 3b). To fur-
ther confirm apoptosis induction, pro-caspase3 and active
caspase-3 levels were detected by western blot analysis.
analyses via trans-well cell migration assay and d quantitative
analysis of migrated cells through trans-well membranes. L929-
CYGB cells failed to migrate from trans-well membranes. Note Scale
bar 200 lm, *p \ 0.05
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138 Mol Cell Biochem (2018) 437:133–142

Fig. 3 Western blot analysis of a PI3K/AKT/mTOR pathway in L929-CYGB cells. CYGB overexpression inhibits phosphorylation
components, b apoptosis-related proteins and their quantitative of mTOR, S6, AKT, and 4E-BP1 indicating the anti-proliferative
measurements. Apoptotic protein expression levels were increased activity. Note *p \ 0.05

Fig. 4 Microarray analyses of for L929-GFP and L929-CYGB cells. a Volcano and b Scatter plot for 29 upregulated and 46 downregulated
genes in L929-CYGB cells. c Signature clusters of genes in L929-GFP and L929-CYGB

While there was not a significant change in pro-caspase 3 overexpressed cells (Fig. 3b). Almost 3-fold higher p53,
levels, active caspase-3 protein expression was high in p21, and active caspase-3 levels were detected in L929-
L929-CYGB cells as a result of apoptosis in CYGB- CYGB cells.

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Mol Cell Biochem (2018) 437:133–142 139

Table 1 Selected genes affected by CYGB overexpression

Gene name Fold difference Function

Upregulated
CADM1 2,29 Cell adhesion, migration, cancer migration [25, 26]
THBS1 1,95 Extracellular matrix formation, inhibition of angiogenesis [27] and wound healing [28]
LDB1 1,77 Focal adhesion and cell migration [29]
Olfr830 1,74 Olfactory sensory neurons [30]
Olfr314 1,63 Olfactory sensory neurons [30]
HABP2 1,62 Tumor suppressor [31], coagulation, and fibrinolysis [32]
Olfr988 1,6 Olfactory sensory neurons [30]
KPTN 1,55 Neuromorphogenesis [33], hearing loss [34]
TRIM8 1,54 Protein ubiquitination and carcinogenesis [35]
Eef1a2 1,53 Cancer metastasis [36], cell invasion, and migration [37]
Zfp628 1,52 Transcription activator
PPARGC1A 1,52 Mitochondrial function and apoptosis [38], cell migration [39]
Downregulated
ADAMTS6 -2,12 Peptidase and metallopeptidase activity, ECM remodeling [40]
SEMA6A -2,01 Neural cell migration [41], cell motility, and proliferation [42]
Cyp4f17 -1,91 Cytochrome P450 family
CSNK1G3 -1,73 Neural cell adhesion [43]
Nfib -1,73 Mesenchymal and epithelial cell proliferation [44]
HSPA9 -1,65 Tumor growth and invasiveness [45]
Tcerg1 -1,61 Cell proliferation [46], apoptosis [47]
PGD -1,59 Tumor cell migration [48]
Pdha1 -1,57 Glycolytic pathway, tricarboxylic cycle
Pfkb3 -1,57 Cell proliferation, apoptosis, and survival in tumor cells [49, 50]
Mex3c -1,56 Growth, energy metabolism [51]
CCR7 -1,55 Dendritic cell migration [52], fibroblast cell proliferation [53]
CSNK1A1 -1,53 Cell proliferation [54]
Neat1 -1,51 Cell proliferation and survival [55]
Note Genes were selected based on ± 1.5-fold difference and p value of less than 0.05

Gene expression profiles of L929 was modified
by CYGB overexpression
To investigate possible gene expression signature in
CYGB-overexpressed cells, gene expression profiles of
CYGB versus GFP control cells were conducted using
microarray analysis. In total, 29 upregulated and 46
downregulated genes were detected in CYGB-overex-
pressed cells as represented by the volcano and scatter plot
reported in Fig. 4a and b. Pathway analysis revealed that
the effected pathways were predominantly related to cel-
lular growth and proliferation, in which 26 genes were
dysregulated (Fig. 4c; Table 1). Remarkably, CYGB
overexpression inhibited some genes crucial for the tumor
growth and invasiveness such as HSPA9 and Pfkb3
(Table 1).
A core set of deregulated 4 genes which have been
reported to negatively regulate cell migration including
CADM1 THBS1 and LDB1 were upregulated in CYGB
overexpression. It was also found that functionally impor-
tant cell proliferation and migration genes were downreg-
ulated significantly in CYGB-positive cells including
ADAMTS6, SEMA6A, Nfib, Tcerg1, PGD, Pfkb3, CCR7,
CSNK1A1, and Neat (Table 1).
Discussion
In the current study, we have shown the negative effects of
CYGB overexpression on migration and proliferation rates
of fibroblast cells through induction of PTEN, repressing
the PI3K/AKT/mTOR pathway and inducing apoptosis.
Forced expression of CYGB inhibited colony-forming
ability, migration, and proliferation rate of fibroblasts. In
line with these findings, migration rate of NIH 3T3
fibroblast were attenuated by CYGB overexpression [20].

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Fig. 5 Potential pathways affected by CYGB overexpression
CYGB-expressing cells were found to express high levels
of p53, p21, and active caspase 3 with respect to control
GFP-expressing cells. CYGB has previously been reported
to be upregulated under genotoxic stress, which was linked
to p53 and p21 accumulations [3]. In the same study,
inhibition of p53 ubiquitination and prolonging half-life of
p53 by CYGB expression were proposed to be the primary
reason for growth inhibition. However, localization and
interactions of p53 and p21 with CYGB have not been
evaluated. Therefore, further studies could clarify the roles
of this unique globin and potential pathways involved in
nuclear fraction.
Microarray gene expression result showed induced
expression of tumor suppressor genes and negative regu-
lators of cell migration such as CADM1, HABP2, THBS1,
and LDB1 upon overexpression of CYGB. Moreover
majority genes downregulated in L929-CYGB cells are
involved in survival, cell proliferation, and migration. In
addition, genes related to extracellular remodeling, tumor
growth, and metastasis were expressed to a much lesser
extent in CYGB-overexpressed cells. These groups of
genes consisted of ADAMTS6, Nfib, HSPA9, PGD, and
Pfkb3 whose expression was downregulated in L929-
CYGB cells.
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Mol Cell Biochem (2018) 437:133–142
Protein analysis in CYGB-induced proliferation and
migration-retarded fıbroblast cells demonstrated that
phosphorylation of positive regulators for the PI3K/AKT/
mTOR pathway including mTOR, AKT, S6, and 4E-BP1
repressed in CYGB-overexpressing cells, while the natural
inhibitor of the AKT pathway, PTEN, was detected to be
upregulated in comparison with the control GFP-express-
ing cells. The most likely explanation for CYGB and PI3K/
AKT/mTOR pathway interaction might be indirect by the
activation of PTEN opposing the activity of PI3K.
Although direct interaction between CYGB and PI3K/AKT
pathway has not been presented before, Xu and co-workers
stated the positive correlation between low expression of
CYGB and PI3K/AKT activation, subsequently leading to
tumor progression in glioma [21]. In addition, elevated
phosphorylation of AKT and extracellular signal-regulated
kinase (ERK) were noticed in livers of CYGB-deficient
mice. These studies and the present study clearly indicate
the strong negative correlation between AKT pathway and
CYGB expression levels (Fig. 5). In addition to CYGB
overexpression, knockdown and/or knockout studies
should strictly be performed as further studies to elucidate
the exact role of CYGB in the regulation of PI3K/AKT/
mTOR pathway in fibroblast cells. As PI3K/AKT pathway
Mol Cell Biochem (2018) 437:133–142
plays critical roles in proliferation and migration [22], and
reduced levels of AKT expression and phosphorylation
have been linked with chronic wounds [23], evaluating
putative CYGB expression in various chronic wounds
would be interesting and might shed new light on the gene
regulations in non-healing wounds. Fibroblast migration
and proliferation is mostly deficient in chronic wounds
[24]. Given the fact that CYGB expression was found to
negatively affect fibroblast migration and proliferation, the
levels of CYGB in chronic wounds such as diabetic wound
ulcers should be investigated to elucidate the potential
contribution of CYGB to the non-healing wound forma-
tion. CYGB could be a new target in clinical dermatology
to increase the regeneration capacity of not only chronic
wounds but also normal acute wounds.
Conclusions
Although many signaling molecules and molecular path-
ways involved in cell motility and proliferation have been
reported in the literature, identification of new genes and
regulators is required to understand not only wound-heal-
ing mechanism but also cancer metastasis. The results of
the current study suggest that CYGB might be a potential
target for non-healing wound therapy. Identification of the
CYGB’s roles in different tissues and in cell behavior
could be valuable for further studies to determine new
approaches for chronic wound healing, cancer, and fibrosis.
Acknowledgements We would like to thank NIH Fellows editorial
board for their assistance for manuscript editing.
Funding This study was supported by Yeditepe University.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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