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Magnesium Research 2013; 26 (1): 24-31
ORIGINAL ARTICLE
Extracellular magnesium and
in vitro cell differentiation:
different behaviour of different cells
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 03/08/2017.
Sara Castiglioni, Marzia Leidi, Elisa Carpanese, Jeanette A.M. Maier
Dipartimento di Scienze Biomediche e Cliniche L. Sacco, Università di Milano, Milano
I-20157, Italy
Correspondence: JAM Maier, Dipartimento di Scienze Biomediche e Cliniche Luigi Sacco, Università di Milano,
Via GB Grassi, 74 Milano, Italy
<[email protected]>
Abstract. The contribution of magnesium to cell differentiation is not clear.
Some studies indicate that low extracellular magnesium promotes cell differentiation, while others reach opposite conclusions. We evaluated the effects
of different concentrations of extracellular magnesium on the differentiation of three in vitro experimental models: human endothelial cells seeded
onto Matrigel; phorbol ester-treated myeloid leukemia U937 cells; and 3T3L1 pre-adipocytes exposed to a hormonal cocktail containing dexamethasone
and insulin.The differentiation of endothelial cells and pre-adipocytes seems
to be independent of extracellular Mg concentration. Conversely, magnesium
deficiency retards, while high extracellular magnesium accelerates phorbol
ester-induced U937 cell differentiation, probably by interfering with calcium
homeostasis or with the activity of kinases.We conclude that the extracellular
magnesium concentration affects the differentiation of various cell types.
Key words: magnesium, cell differentiation, endothelial cells, pre-adipocytes, U937
cells
is peroxisome proliferator-activated receptor-␥
(PPAR␥) [3]. PPAR␥ induces the expression of
adipocyte-specific genes through the binding
of PPAR␥-retinoid X receptor heterodimers
to a PPAR-response element, resulting in the
promotion of intracellular fat storage.
Endothelial differentiation is a fundamental
process for the development of the vasculature
and for angiogenesis [4]. A convenient model
for studying endothelial cell differentiation in
vitro is the tube formation assay on Matrigel, a
reconstituted basement membrane extracellular
matrix [5]. When plated onto Matrigel, endothelial
cells rapidly attach, align, and form capillarylike tubules which contain a lumen and tight
cell-cell contacts. Both transcription-dependent
and -independent mechanisms are involved in
endothelial cell tube formation on Matrigel.
Recently, gene array experiments have shown that
during Matrigel-induced tube formation, human
24
To cite this article: Castiglioni S, Leidi M, Carpanese E, Maier JAM. Extracellular magnesium and in vitro cell differentiation:
different behaviour of different cells. Magnes Res 2013; 26(1): 24-31 doi:10.1684/mrh.2013.0330
doi:10.1684/mrh.2013.0330
While clear evidence has been provided about
the relevance of magnesium (Mg) in driving cell
proliferation, little is known about its participation to the modulation of cell differentiation
[1]. Differentiation, crucial during all stages of
life, from embryo to adult, is triggered by soluble factors, such as growth factors, cytokines and
hormones, or by signaling from components of
the extracellular matrix, all activating the transcription of genes that control the acquisition of
a differentiated phenotype. The genetic program
activated in response to pro-differentiating signals is cell-specific.
For instance, the differentiation of preadipocytes into adipocytes is regulated by a
complex network of transcription factors that
control the expression of hundreds of genes
responsible for establishing the mature adipocyte
phenotype [2]. The main protein orchestrating
the differentiation and function of adipocytes
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 03/08/2017.
Extracellular magnesium and in vitro cell differentiation
endothelial cells from the umbilical vein (HUVEC)
up-regulate the expression of 86 genes and downregulate the expression of 65, mainly associated
with cell proliferation and metabolism [6]. By subtractive cDNA cloning, the cytoskeletal protein
thymosin ␤4 has been found to be up-regulated
early after seeding onto Matrigel [5]. Interestingly,
in HUVEC transfected with thymosin ␤4, the rate
of tube formation on Matrigel was accelerated,
while an antisense oligomer against thymosin ␤4
inhibited tube formation on Matrigel [5].
Non-adherent, autonomously proliferating
myeloid leukemia cells, including the human
U937 cell line can be stimulated to differentiate
along the monocyte/macrophage pathway by
treatment with 12-O-tetradecanoyl-phorbol-13acetate (TPA) [7]. Indeed, in response to TPA,
U937 cells become adherent, are growth-arrested
and undergo several morphological and functional changes typical of normal monocytes and
macrophages [7].
We asked whether different extracellular
concentrations of Mg might affect cell differentiation. To this end, we selected the three
aforementioned experimental models: HUVEC
seeded onto Matrigel; TPA-treated myeloid
leukemia U937 cells; and 3T3-L1 pre-adipocytes
induced to differentiate into adipocytes.
Methods and materials
Cell culture and differentiation
For all of the cells studied, an Mg-free medium
(Life Technologies Ltd, Paisley, United Kingdom) was used to vary the concentrations of
Mg by adding MgSO4 . On the basis of previous reports [8, 9], we cultured cells in medium
containing 0.1, 1.0 or 5.0 mM Mg; 1.0 mM Mg
served as the control physiological concentration.
Murine 3T3-L1 pre-adipocytes were maintained
in DMEM containing 10% calf serum for two days
post-confluence. Differentiation was then induced
(day 0) in medium containing different concentrations of Mg supplemented with 10% fetal bovine
serum (FBS) (Euroclone, Milan, Italy), 0.5 mM 3isobutyl-1-methylxanthine, 1 ␮M dexamethasone,
and 10 ␮g/mL insulin. After 48 h, the differentiating medium was removed and the cells were
maintained in the corresponding medium containing different concentrations of Mg with 10%
FBS. The medium was changed every two days.
Oil Red O (Sigma Aldrich, Milan, Italy) staining was performed to detect intracellular lipids
[10]. The cells were photographed using a phasecontrast microscope. For a quantitative assay, Oil
Red O dye was extracted with isopropanol 100%
and absorbance was measured at 500 nm by spectrophotometry.
Primary HUVEC isolated from the umbilical
vein were cultured in M199 containing 10% FBS,
1 mM glutamine, endothelial cell growth factor
(150 ␮g/mL), 1 mM sodium pyruvate and heparin (5 units/mL) on 2% gelatin-coated plates.
Tube formation on Matrigel was evaluated as
a model to study endothelial differentiation [5].
Culture plates were coated with cold Matrigel (BD
Bioscience, Buccinasco, Italy) and incubated for
30 minutes at 37◦ C. HUVEC were cultured for
36 h in media containing 0.1, 1.0 and 5.0 mM Mg.
The cells were then seeded in their corresponding
medium onto Matrigel and photographed using
a phase-contrast microscope after different time
intervals.
Human myeloid leukemia cell line U937 cells
were grown in RPMI 1640 medium containing
10% FBS and 2 mM glutamine. After 72 h of
culture in medium containing different concentrations of Mg, the cells were treated with 25 nM
TPA (Sigma, St. Louis, MO, USA) for 12, 24,
48 and 72 h. To measure plastic adherence, the
supernatant was decanted, adherent cells were
harvested with a rubber policeman, and the total
number of attached cells was determined using a
hemocytometer.
Western blot
Western blot was performed on cell lysates.
Proteins were separated on 12% SDS-PAGE,
transferred onto nitrocellulose sheets at 200 mA
overnight, and probed with anti-PPAR␥ 1-2,
anti-p21 and anti-actin antibodies (from Santa
Cruz Tebu-bio, Magenta, Italy). The SuperSignal chemiluminescence kit (Pierce, Rockford, IL,
USA) was used to detect immunoreactive proteins.
All of the experiments were repeated at least three
times with comparable results.
RT-PCR
HUVECs were lysed in 1 mL Trizol and RNA was
purified, cDNA was synthesized from 1 ␮g of total
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S. CASTIGLIONI, ET AL.
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RNA using oligo(dN)6 primers using the Transcriptor first-strand cDNA synthesis kit (Roche
Diagnostics, Basel, Switzerland). PCR amplification was performed as follows: 45 sec at 94◦ C, 45
sec at 60◦ C, and 1 min at 72◦ C. The reaction was
stopped after 35 cycles. The primers for thymosin
␤4 were: 5’-CAGACC AGACTTCGCTCGTA-3’
and
5’-GCTTCTCCTGTTCAATCGT-3’.
RTPCR with specific primers for GAPDH was
performed to normalize (sense 5’- CCACCCATGGCAAATTCC
ATGGGA-3’;
antisense
5’-TCTAGACGGCAGGTCAGGTCCACC-3’).
Statistical analysis
Statistical significance was determined using the
t test and set at p values at 0.01.
Results
HUVEC differentiation on Matrigel
Under normal culture conditions, HUVECs
seeded onto Matrigel attach within one h. Then,
in four-six hours, the cells align and form lateral
associations which progress to the formation of
tubes over the next 8-10 hours [11].
After 36 h of culture in media containing 0.1, 1.0
or 5.0 mM Mg, HUVEC were seeded onto Matrigel
and maintained in their corresponding medium.
Figure 1A shows no difference in cell organization in HUVEC cultured in 0.1, 1.0 and 5.0 mM
Mg at any of the time points evaluated. We also
studied the expression of thymosin ␤4, which is
known to be rapidly upregulated in HUVEC on
Matrigel [5]. As shown in figure 1B, we detected
thymosin ␤4 only in HUVEC seeded onto Matrigel
for 4 h with no differences related to the various
concentrations of Mg used to culture the cells.
3T3-L1 pre-adipocyte differentiation in
vitro
To explore whether different concentrations of Mg
affect adipogenesis, 3T3-L1 cells were exposed to
medium containing 0.1, 1.0 or 5.0 mM Mg and
induced to differentiate with a hormonal cocktail
up to day 8. At different time points, we stained
the cells with Oil Red O to visualize the presence
of lipid droplets in the cytosol. We did not observe
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any difference between 3T3-L1 cultured in different concentrations of Mg (figure 2A), and this
was confirmed by spectrophotometry (figure 2B).
We then investigated whether the expression of
PPAR␥ is modulated during the differentiation
of 3T3-L1 cells cultured in 0.1, 1.0 or 5.0 mM
Mg. The total amounts of PPAR␥ were evaluated at the beginning of the experiment (day 0)
and after exposure to the hormonal cocktail for 4
and 8 days. Western blot revealed that the levels of PPAR␥ markedly increased 4 days after
the treatment with the differentiation cocktail,
independently from the extracellular concentration of Mg (figure 2C). After 8 days, when the cells
had reached terminal differentiation, PPAR␥ levels were decreased.
Myeloid leukemia U937 cell
differentiation in vitro
To study the effect of different concentrations
of extracellular Mg on macrophage differentiation, we used the differentiation system of human
myelocytic U937 cells [12], which can be induced
to differentiate into adherent macrophage-like
cells by phorbol esters [13]. In parallel, cell cycle
arrest occurs. We exposed the cells to TPA and
counted the number of attached cells. TPA clearly
induced U937 cell differentiation with a pronounced increase in adherent cells within the
first 24 h. Our results show that high concentrations of extracellular Mg accelerated, while
low Mg retarded TPA-induced U937 adhesion to
plastic (figure 3A). We also investigated the levels of the cyclin-dependent kinase inhibitor p21,
which is induced after 24 h exposure of U937
cells to TPA. Figure 3B shows that p21 is undetectable in untreated cells and is upregulated by
TPA. Interestingly, the total quantity of p21 is
less pronounced in TPA-treated U937 cells under
Mg restriction than controls. No significant differences were observed in p21 levels between
U937 cells in 1.0 and 5.0 mM Mg-containing
medium.
Discussion
Differentiation is orchestrated by a variety of
influences, such as growth factors, hormones,
cell-cell contacts and cell-extracellular matrix
Extracellular magnesium and in vitro cell differentiation
A
Mg (mM)
0.1
1.0
5.0
20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
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1h
4h
8h
B
Mg (mM)
Matrigel
1.0
0.1
-
+
-
5.0
+
-
+
thymosinβ4 actin -
Figure 1. The effect of different concentrations of Mg on HUVEC differentiation. HUVEC were cultured for 36 h in 0.1, 1.0 or 5.0 mM Mg before being seeded onto Matrigel. A) Photographs were taken
1, 4 and 8 h after seeding onto Matrigel. B) RT-PCR using specific primers was performed after 4 h on
Matrigel. GAPDH was used to show that comparable amounts of RNA were used per lane.
interaction. Even though Mg is a versatile electrolyte that has been shown to be involved in many
cellular processes [14], the relationship between
Mg and cell differentiation remains unclear.
Mg-restricted media rapidly reversed the transformed phenotype of fibroblasts, thus suggesting
that Mg promotes pro-differentiating events [15].
In C2C12 myogenic cells, Mg deficiency upregulated Myod, one of the transcription factors
fundamental for myogenesis [16].
Conversely, in pancreatic islet RINm5F cells,
both low and high extracellular Mg impaired
insulin synthesis without altering cell morphology [17]. Furthermore, Mg deficiency reversibly
antagonized the differentiation of promyelocytic
leukemia HL60 cells exposed to differentiating
agents such as DMSO and retinoic acid [18]. Our
results on U937 cells are in agreement with this
finding. U937 cells normally grow in suspension,
but upon exposure to TPA they extend pseudopodia and became adherent to each other and to
the surface of the culture dish. We observed that
Mg restriction retards TPA-induced cell differentiation. To this end, since i) p21 is a primary
27
S. CASTIGLIONI, ET AL.
A
Mg (mM)
0.1
1.0
5.0
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
Day 4
Day 8
B
0,6
Absorbance (OD 500 nm)
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Day 0
0,5
0,4
0,3
0,2
0,1
0
Day 4
Day 0
0.1 mM Mg
C
Day 0
Mg (mM)
0.1
1.0
Day 8
1.0 mM Mg
5.0 mM Mg
Day 4
5.0
0.1
1.0
Day 8
5.0
0.1
1.0
5.0
PPARγ actin -
Figure 2. The effect of different concentrations of Mg on 3T3-L1 pre-adipocyte differentiation. 3T3-L1
cells were induced to differentiate in 0 1, 1.0 and 5.0 mM Mg for 4 and 8 days. A) The cells were stained
with Oil Red O after 4 and 8 days of differentiation, and photographed (20× magnification). B) Oil Red
O was extracted with isopropanol and absorbance measured at 500 nm. C) Cell lysates (70 ␮g) from
3T3-L1 cells, induced to differentiate, were separated by SDS-PAGE. Western blot was performed with
antibodies against PPAR␥. The blot was incubated with an anti-actin antibody to show that comparable
amounts of protein were loaded per lane.
28
Extracellular magnesium and in vitro cell differentiation
Adherent cells (number)
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A
120000
*
100000
*
80000
60000
*
40000
*
20000
0
12h
24h
0.1 mM Mg + TPA
B
48h
1.0 mM Mg + TPA
+ TPA
Mg (mM)
0.1
1.0
72h
5.0 mM Mg + TPA
- TPA
5.0
0.1
1.0
5.0
p21 -
actin -
Figure 3. The effect of different concentrations of Mg on U937 cell differentiation.
The cells were cultured in medium containing different concentrations of Mg for 72 h and then treated
with TPA (25 nM) for different times. A) After 12, 24, 48 and 72 h in the presence of TPA, adherent cells
were harvested with a rubber policeman and counted. Data are shown as the mean ± standard deviation
(*p<0.01). B) Cell lysates (70 ␮g) from U937 cells induced to differentiate for 24 h, were separated by
SDS-PAGE. Western blot was performed with antibodies against p21. The blot was incubated with an
anti-actin antibody to show that comparable amounts of protein were loaded per lane.
response gene strictly related to the early differentiation program of many hematopoietic cell
lines including U937 cells [19] and ii) p21 plays
a role in facilitating U937 differentiation [20], it
is worth noting that the levels of p21 are lower
in TPA-treated cells under Mg restriction than in
cells cultured in 1.0 or 5.0 mM Mg. The induction
of p21 in U937 cells by TPA is p53-independent
and mediated through the activation of protein
kinase C (PKC)/MAPK pathways [21]. Considering that the transphosphorylation from ATP
is Mg-dependent, it is likely that Mg deficiency
might impair the aforementioned pathways. Alter-
natively, since Mg acts as an intracellular second
messenger, it is possible that Mg restriction
impairs transient Mg influx necessary for several
cell activities. While no differences were observed
in p21 levels between cells cultured in 1.0 versus 5.0 mM Mg, our results indicate that high
extracellular Mg accelerates U937 cell differentiation. This could be due to the well known
antagonisms between Mg and calcium leading to
an imbalance in the intracellular Mg/calcium ratio
with consequent alterations of calcium-mediated
signals, and/or to a potentiation of transphosphorylation reactions. All together, these data
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S. CASTIGLIONI, ET AL.
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concerning Mg and leukemia cell differentiation
suggest that controlling and correcting Mg homeostasis in leukemic patients might be important
for better outcomes.
Recently, we have shown that excessive extracellular Mg blocks pre-osteoblast differentiation
[8], which is in agreement with a previous report
showing that high extracellular Mg inhibited
matrix mineralization in the prechondrogenic cell
line ATDC5 [22]. The contrasting results about the
effect of high Mg on the differentiation of U937
cells on one side and condrocytes and osteoblasts
on the other are not surprising, since differentiation requires the activation of cell-specific genetic
programs that can be differentially affected by
Mg.
The differentiation of endothelial cells and
pre-adipocytes seems to be independent from
extracellular Mg. Indeed, we detected no effects of
different concentrations of Mg in HUVEC induced
to differentiate on Matrigel. Accordingly, we found
no alteration in the expression of thymosin ␤4,
an early gene induced by culture on Matrigel.
Thymosin ␤4 is involved in the differentiation of
many cell types, including HUVEC [5]. Since the
reorganization of actin filaments into fiber bundles is essential for tube formation on Matrigel,
it is interesting that thymosin ␤4 is a G-actinsequestering protein directly implicated in the
cytoskeletal remodeling required for tube formation [5].
We did not observe any change in the accumulation of lipid droplets or in the extent of
PPAR␥ upregulation in pre-adipocytes cultured in
different concentrations of Mg and induced to differentiate with a hormonal cocktail. Our results,
however, are in disagreement with a previous
report demonstrating depressed differentiation in
primary porcine stromal-vascular cells induced
to differentiate into adipocytes in Mg-deficient
medium [23]. The contrasting results can be
ascribed to the completely different experimental
model used.
Together, these findings suggest that fundamental differences exist between various cell types in
the contribution of Mg to cell differentiation.
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