International Journal of Women’s Health
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Diet-induced vitamin D deficiency triggers
inflammation and DNA damage profile in murine
myometrium
Heba elhusseini 1
Hoda elkafas 1,2
Mohamed abdelaziz 3
sunil Halder 1
Ihor atabiekov 1
noura eziba 1
nahed Ismail 4
abdeljabar el andaloussi 1
Ayman Al-Hendy 1
1
Department of Obstetrics and
Gynecology, University of Illinois
of Chicago, Chicago, IL, USA;
2
Pharmacology and Toxicology
Department, National Organization
for Drug Control and Research, Cairo,
Egypt; 3Department of Obstetrics
and Gynecology, Mansoura University
Hospital, Mansoura Faculty of
Medicine, Mansoura, Egypt; 4clinical
Microbiology Division, University of
Illinois of Chicago, Chicago, IL, USA
Background: Previously, we reported a significantly higher prevalence of uterine fibroids
(UFs) in African American women. This minority group also commonly suffers from vitamin D
deficiency. We have demonstrated that 1,25(OH)2D3 attains a fibroid growth inhibitory impact
through its ability to block the G1/S (gap 1/synthesis) phase of the cell cycle. Vitamin D is
involved in DNA damage as well as in immune response regulation, anti-inflammation, autoimmunity and cancer. Since most of the prior data on vitamin D and UF were generated in vitro
via established cell lines, it was necessary to verify and validate this observation in vivo using
a diet-induced vitamin D-deficient mouse model.
Materials and Methods: Our model of vitamin D lacking function was established using
8-week exposure of C57/BL6 mice to vitamin D-deficient diet provides evidence of different
functions accomplished by vitamin D in the regulation of myometrium homeostasis disrupted
in the context of uterine fibroid.
Results: We found that vitamin D deficiency was associated with increased expression of sex
steroid receptors in murine myometrium, increased expression of proliferation related genes,
the promotion of fibrosis and enhanced inflammation, and promoted immunosuppression
through Tregs expansion in murine myometrium. We also showed that a vitamin D deficient
diet enhanced DNA damage in murine myometrium.
Conclusion: Our model mimics the effects in humans that a lack of vitamin D has and propels
the study of in vivo interaction between inflammation, genomic instability and cell proliferation
in the myometrium.
Keywords: vitamin D, diet, inflammation, DNA damage, uterine fibroids
Introduction
correspondence: abdeljabar el
Andaloussi; Ayman Al-Hendy
909, south Wolcott avenue, 7111
College of Medicine Research Building
(MC 808), University of Illinois at
Chicago, Chicago, IL 60612, USA
Tel +1 312 996 5629
email aelandal@uic.edu;
aalhendy@uic.edu
Uterine fibroid (UF) is a serious health disparity as they are three to four times more
prevalent in women of color compared to Caucasian women. Although there are
many factors associated with an increased risk of UF development, such as nulliparity, early menarche, obesity and genetics, the race is considered to be one of
the most important among them. We have reported that the estimated prevalence of
UFs remains significantly higher in African-American women even after controlling for potential confounders such as parity and body mass index.1 These minority
women also commonly suffer from vitamin D deficiency.2 There are limited dietary
sources rich in vitamin D, such as fatty fish, fish oils and fortified foods; however,
sunlight exposure remains the main source of vitamin D.3 That is likely why darkskinned individuals commonly suffer from vitamin D deficiency because their skin
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http://dx.doi.org/10.2147/IJWH.S163961
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elhusseini et al
contains more melanin that equates to greater protection
against ultraviolet radiation exposure.4,5
Additionally, we have shown recently that vitamin D
inhibits in vitro UF cell proliferation by modulating multiple
cellular pathways such as downregulation of catechol-Omethyltransferase (COMT), a key enzyme for clearance of
catechol estrogens.6 Furthermore, our previous data showed
that vitamin D3 suppressed TGF-β3-induced fibrosis,
Wnt/β-catenin and mTOR as well as sex steroid receptors’
gene expression in human fibroid cells.6–9 We have also
demonstrated that 1,25(OH)2D3 attains an inhibitory impact
on fibroid growth through its ability to block the G1/S (gap
1/synthesis) phase of the cell cycle. Moreover, it regulates
several cell cycle regulatory genes and modulates activities
of cyclin-dependent kinases, leading to a reduced number
of cells in the S phase and thus their accumulation in the
G0–G1 phase.10,11
The association of vitamin D and vitamin D receptor
(VDR) with DNA repair has been examined in the model
of Ras-induced senescence. A functional relationship was
demonstrated between the Ras oncogene, legend-mediated
stability of VDR axis and the expression of DNA repair
factors.12 Indeed, deficiency in vitamin D can create genomic
instability that promotes tumorigenic transformation.13–15
Vitamin D is involved in immune response regulation,
prevention of inflammation and autoimmunity, which are
major risk factors for cancer progression.15–17 VDR exerts
its anti-inflammatory effect via downregulation of NF-kBdependent production of proinflammatory cytokines and
chemokines.18 The anti-inflammatory effect of vitamin D is
also critical for maintaining tissue homeostasis. In addition,
vitamin D plays a role in activation and differentiation of
T cells as well as regulation of autophagy and antibacterial
function of monocytes and macrophages.19–21 Recently, we
reported that autophagy is abrogated in human UFs due
to defective expression in Atg4D.22 Based on studies by
us and other investigators, vitamin D deficiency is poised
to be an important risk factor for myometrial transformation secondary to chronic inflammation that likely leads to
genomic instability and accumulation of abnormalities in the
DNA repair machinery.
Since most studies have examined the effect of vitamin D
deficiency on fibroid development using in vitro cell lines,
we used an in vivo mouse model with vitamin D-deficient
diet to verify and validate the effect of vitamin D deficiency
on inflammation, genomic instability and proliferation of
myometrium. This model recapitulates myometrial changes
in humans with vitamin D deficiency, and thus, data from
this study can have a high translational impact.
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Materials and methods
Induction of vitamin D deficiency in mice
Female C57BL/6 mice, 4–6 weeks old, were divided into
two groups: control group (n = 10) fed on normal diet
(Purina #5053) containing vitamin D of 4 IU/g versus vitamin
D-deficient diet group (n = 10; Purina #5B7Q) totally lacking
vitamin D for 8 weeks. The diet was obtained from Charles
River Laboratories. All animal procedures were conducted in
accordance with the National Institutes of Health Guidelines,
and mice were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal
Care. All animal experiments were approved by the animal
committee of Augusta University Institutional Animal Care
and Use Committee (IACUC). At the end of the experiment,
all animals were euthanized. Blood was collected by cardiac
puncture under inhalation anesthesia prior to euthanasia to
measure serum vitamin D and estrogen (E2). Myometrial
tissues were collected from two groups, the endometrium
was opened, scrubbed and divided into two samples, one was
immediately snap frozen in liquid nitrogen and then stored
at −80°C and the other was fixed in 10% buffered formalin
for immunohistochemistry.
Immunohistochemistry
Paraffin-embedded tissue sections (4 µm) were deparaffinized
and rehydrated by passing through xylene and graded ethanol
solutions as previously described.23 Sex steroid receptors were
evaluated by staining with progesterone receptor A (PRA)
and progesterone receptor B (PRB) and ER-alpha (ER-α).
Cell proliferation was analyzed by staining with immunoreactive proliferating cell nuclear antigen (PCNA) and a cell
proliferating marker Ki-67. Apoptosis in myometrial tissues
was evaluated using histochemical staining with anti-caspase
3 antibody. Fibrosis-related receptors were evaluated by
using TGFβR2. DNA damage was evaluated by staining with
immunoreactive DNA damage marker γH2AX, RAD50 and
RAD51. For the quantification, we compared the two groups
(control group and vitamin D3-deficient group) by taking 10
fields of each, counting the number of positive cells (brown
staining) and calculating the percentage of positivity of each
antibody by dividing the brown nucleus by the total numbers
of the nucleus in one field and then taking the average.
Western blot
Frozen myometrial tissues were homogenized and sonicated
in sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) lysis buffer. Preparation of protein lysates and
Western blot analyses were performed as previously described
by Halder et al.24 Briefly, the crude lysates were centrifuged
International Journal of Women’s Health 2018:10
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at 13,000 rpm for 20 min at 4°C. The clear protein lysates
were transferred into new Eppendorf tubes, and protein
concentrations were determined using Bio-Rad protein assay
reagents according to the manufacturer’s instructions. Equal
amounts of each protein lysate (30–40 µg) were resolved by
10% SDS-PAGE and transferred onto the polyvinylidene
fluoride membrane. Western blot analyses were performed
to assess the expressions of sex steroid receptors using antiPR-A (1:300), anti-PR-B (1:300) and anti-ER-α (1:500); cell
growth and proliferation markers using anti-PCNA (1:500)
and anti-cyclin D1 (1:500); and apoptotic markers using
anti-PARP (1:500) antibodies. Fibrosis-related markers were
evaluated using TGFβR2 (1:500). The antigen–antibody
complex was detected using an enhanced chemiluminescence
detection system (Amersham Bioscience, Piscataway, NJ,
USA). Specific protein bands were visualized after exposure
to autoradiography films and development using an automatic
X-ray developer. Sample protein quantification of Western
blot data was calculated as a ratio to actin level via densitometry. Densitometry analysis was performed using Image
Studio software (Lincoln, NE, USA).
Flow cytometry
The infiltrating immune cells of the myometrium were stained
for the common marker CD45. The regulatory T cells (Tregs)
were stained by their specific markers CD4 and Foxp3. The
quantification of suppressive cytokines released by Tregs was
performed based on TGF-β and IL-10 intracellular staining after
fixation and permeabilization using Fix/Perm kit (BD Biosciences, San Jose, CA, USA). The intracellular staining for cytokines was done in the presence of GolgiStop (BD Biosciences).
A total of 1 × 105 cell line cells were incubated with the Abs
for 30 min at room temperature. The cells were washed twice
with PBS/2% fetal bovine serum (FBS) (v/v) and re-suspended.
Samples were acquired and analyzed using the FACSCanto (BD
Biosciences) and FlowJo systems (Ashland, OR, USA).
Real-time polymerase chain reaction (PCR)
Total RNAs were isolated by using TRIzol reagent (Thermo
Fisher Scientific, Waltham, MA, USA) according to the
manufacturer’s instructions. RNA was extracted from
frozen myometrium tissue. A total of 1–5 µg of total RNA
was reverse transcribed using SuperScript III Reverse
Transcriptase (Thermo Fisher Scientific) to prepare cDNA.
Real-time PCR was performed by using iTaq Universal SYBR
Green Supermix (Bio-Rad Laboratories Inc., Hercules, CA,
USA) for murine TGF-β, IL-10 and β-actin using specific
primers (Table S1). The amplification was done on Bio-Rad
quantitative polymerase chain reaction (qPCR) machine
International Journal of Women’s Health 2018:10
role of vitamin D in uterus
(Bio-Rad Laboratories Inc.). The results are represented as
an expression relative to actin.
Radioimmunoassay (RIA)
Sera were analyzed for E2 using the RIA method previously
validated in our laboratory.25 The sensitivity of E2 assay
was 2 pg/tube, and the intra- and inter-assay coefficients of
variation (CVs) were 4.9% and 10.8%, respectively. Sera
were also analyzed for vitamin D (25 hydroxy vitamin D)
level. The vitamin D assay involves a rapid extraction of
vitamin D from serum or plasma with acetonitrile. The
extracted sample was assayed using an equilibrium RIA procedure based on an antibody that is co-specific for vitamin D.
The assay has a range of 2.5–100 ng/mL and intra- and interassay CVs of 8.0% and 10.0%, respectively. Measurement of
vitamin D was done at Heartland Assays (Ames, IA, USA)
and that of E2 was done at Ligand Assay and Analysis Core
Laboratory (Charlottesville, VA, USA).
Statistical analyses
All statistical analyses were performed using paired t-tests.
The paired t-test was used to assess the significance of differences between the two groups. Values were considered to
be statistically significant when P was ,0.05.
Results
Vitamin D-deficient diet effectively
induces vitamin D deficiency in mice
Our experimental design aimed to induce in vivo vitamin D
deficiency through a vitamin D-deficient diet (Figure 1A).
At 8 weeks after feeding, we measured the serum levels of
vitamin D in both groups using the standard RIA technique
as described before.25 We observed that the serum level of
vitamin D in the deficient group was significantly decreased
(P , 0.05) compared to the control group (Figure 1B).
Decreased levels of vitamin D in mice did not affect serum
estradiol level as reported previously,26,27 and there was
no significant difference between vitamin D-sufficient
and -deficient mice (Figure 1C). After 8 weeks of mice exposure to vitamin D-deficient diet, the mice appeared healthy
with normal uterus weight compared to the control group
with the absence of morbidity or mortality.
Vitamin D deficiency is associated with
increased expression of sex steroid
receptors in murine myometrium
We were then set to examine if vitamin D deficiency induces a
pre-fibroid status in the myometrium of affected mice. To this
end, we examined several parameters that are characteristic of
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Notes: (A) Study design of mice exposition to deficient vitamin D diet for a time period of 8 weeks. Mice serum level of vitamin D (B) and estradiol D3 (C) from vitamin D
deficiency and control groups (n = 10) were measured using RIA (*P # 0.05).
Abbreviation: Vit, vitamin.
fibroid tumors (ie, increased sex steroid receptor expression
and proliferation, decreased apoptotic cell death, increased
production of extracellular matrix and enhanced inflammation as well as DNA damage). Using Western blot, we first
examined the effect of vitamin D deficiency on the levels
of sex steroid receptor expression in the myometrium using
tissues collected from the two groups. Our results showed
a significant increase in PRA (P , 0.05), PRB (P , 0.05)
and ER-α (P , 0.05) expression levels in the vitamin D
deficiency group compared to the control group, semiquantified by densitometry (Figure 2A and B). These data
were supported by immunohistochemistry using specific antimouse antibodies against PRA, PRB and ER-α antibodies.
Antibody-positive stained cells were much more abundant in
the vitamin D-deficient group when compared to the control
group, and the difference was highly significant, especially
for PRB (P , 0.005; Figure 2C and D).
Vitamin D deficiency increases expression
of proliferation-related genes PCNA and
cyclin D1 in murine myometrium
To examine the effects of vitamin D deficiency on the basal
level of myometrial cell proliferation in affected mice, we
analyzed expression of proliferation gene, PCNA and cell
cycle marker, cyclin D1, by Western blot analyses using
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myometrial tissues from the two groups. We found that
vitamin D deficiency significantly increased the protein
expression of PCNA and cyclin D1 genes compared to
healthy controls (P , 0.05; Figure 3A and B). Consistent
with the immunoblot data, immunohistochemistry staining
demonstrated significantly a higher percentage of PCNA
positive cells and Ki-67+ cells (marker of cell proliferation)
in the treated group when compared to the control group
(P , 0.05; Figure 3C and D).
Vitamin D deficiency decreases the
expression of apoptosis-related genes in
murine myometrium
Decreased apoptotic cell death is an important feature of
UF lesions. We investigated here the impact of vitamin D
deficiency on the apoptosis of myometrial cells in affected
mice by comparing the levels of proapoptotic genes PPAR
and caspase-3 in myometrium. Immunoblot analysis showed
a significant inhibition of PARP in the myometrial tissues
of vitamin D-deficient mice when compared to healthy
controls (P , 0.05; Figure 4A). In addition, immunohistochemistry analysis demonstrated a significantly lower
expression of caspase-3 (P , 0.05; Figure 4B) in the myometrial tissues of vitamin D-deficient mice when compared
to healthy controls.
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Notes: (A) Western blot analyses of myometrial cell lysates used to prepare proteins screened with antibodies against PRA, PRB, ER-α and β-actin. (B) Protein expression was quantified by densitometry based on the matched actin
expression. (C) Immunohistochemistry staining of embedded fixed myometrium isolated from both groups of mice was prepared as described in the “Materials and methods” section and probed specific mouse anti-PRA, anti-PRB and anti-ER-α.
(D) The percentage of positive cells stained compared to that of the controls exposed to regular diet with vitamin D is represented in graph bars. Yellow arrows point to the positive nuclei (n = 10, *P # 0.05, **P # 0.005, ***P # 0.0005).
Abbreviations: PRA, progesterone receptor A; PRB, progesterone receptor B; ER-α, ER-alpha; Vit, vitamin.
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Notes: (A) Western blot analyses with mouse anti-PCNA, anti-cyclin D1, marker of cell cycle and anti-β-actin. (B) Protein densitometry analysis was done as described
in the Materials and methods section. (C) Immunohistochemical analysis of myometrial tissues of vitamin D deficiency and control groups stained by anti-PCNA and antiKi-67. (D) The number of positive stained cells (brown nuclei). Black arrows point to the positive nuclei. All images were captured at 63× magnification, scale bar 200 µm)
(*P # 0.05).
Abbreviations: PCNA, proliferating cell nuclear antigen; Vit, vitamin.
Vitamin D deficiency increased
expression of fibrosis-related genes
Vitamin D supplementation in vitamin D-deficient women
with reproductive disorder significantly decreases the
bioavailability of TGF-β1.28 To test the effect of vitamin D
deficiency on fibrosis-related receptor expression, we performed Western blot analysis using myometrial tissues from
two groups. We observed that the expression level of TGFβR2
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was significantly increased in the vitamin D deficiency group
compared to healthy controls (P , 0.05; Figure 4C). These
data were supported by immunohistochemistry analysis
(P , 0.005). TGFβR2 receptors were expressed at higher
levels in the nuclei of myometrium cells in vitamin D-deficient
group compared to control group (Figure 4D). These data
suggest that vitamin D is antifibrogenic as it modulates the
expression of TGFβR2, a major pro-fibrotic mediator.29
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Figure 4 Vitamin D deficiency inhibits apoptosis and enhances TGFβR2 in myometrium.
Notes: (A) Western blot of PARP using mouse anti-PARP antibody. (B) TgFβR2 Western blot using mouse anti-TGFβR2 antibody supported by quantification analysis by
densitometry. (C) Immunohistochemical analysis for apoptosis marker caspase-3 using mouse anti-caspase-3. (D) Immunohistochemical analysis for fibrosis in myometrium
tissue using mouse anti-TGFβR2. All images were captured at 63× magnification, scale bar 200 µm. The number of stained cells (brown nuclei) was counted in five random
high power fields, and the average number of cells is presented in bar graph. Yellow arrows point to the stained nuclei (n = 10, *P # 0.05, **P # 0.005).
Abbreviation: Vit, vitamin.
International Journal of Women’s Health 2018:10
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elhusseini et al
Vitamin D deficiency enhances
inflammation and promotes
immunosuppression through expansion
of Tregs in murine myometrium
Vitamin D is an important player in the regulation of
inflammation. We hypothesized that vitamin D deficiency
may impair infiltration and/or expansion of Tregs in the
myometrium, which in turn could lead to uncontrolled
inflammation. To test this hypothesis, we examined the
frequency of effectors and Tregs in the myometrium using
flow cytometry. Cells were stained with anti-CD45 (markers
of mononuclear cells) antibody, and CD45+ cells were gated
and further analyzed for expression of Foxp3 (intracellular
transcription factor in Tregs; Figure 5A and B). Our data
show a significant increase in the percentage and mean fluorescence intensity (MFI) of infiltrating CD45+Foxp3+ cells in
the myometrium from vitamin D-deficient group compared
to the healthy control group (P , 0.05). As the suppressive
function of Tregs is defined based on their production of
suppressive cytokines of immune response such as TGF-β
and IL-10, we analyzed the percentage of Tregs expression
in TGF-β and IL-10. Our data show that the vitamin D-deficient group has a significantly higher percentage of Tregs
producing TGF-β and IL-10 than that detected in the healthy
control group (Figure 5C). Furthermore, we examined the
expression of TGF-β and IL-10 mRNA in the myometrium
by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and detected significantly increased levels
of TGF-β and IL-10 expression in the vitamin D deficiency
group compared to the healthy control group (P , 0.05),
which is consistent with the fluorescence-activated cell sorting (FACS) data (Figure 5D and E). Together, these data
suggest that vitamin D deficiency alters the tumor microenvironment in a manner that promotes inflammation.
Vitamin D deficiency enhanced DNA
damage in murine myometrium
Under the absence of vitamin D, the myometrium milieu
becomes proinflammatory and promotes the conversion and
expansion of Tregs from naïve CD4 T cells, which is signature
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Figure 5 Role of vitamin D in the immune homeostasis of myometrium milieu.
Notes: (A) Flow cytometry analysis of myometrium total CD45+ immune cells’ infiltration. (B) MFI analysis after intracellular staining of Foxp3+ Tregs marker gating on
cD45+cD4+ T cells. (C) Percentage of positive Tregs for TGF-β1 (upper panels) and IL-10 (lower panels) based on intracellular staining and FACS analysis with MFI of
(D) TgF-β1 and (E) IL-10, respectively (n = 10, *P # 0.05).
Abbreviations: MFI, mean fluorescence intensity; Tregs, regulatory T cells; Vit, vitamin.
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International Journal of Women’s Health 2018:10
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role of vitamin D in uterus
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Figure 6 Vitamin D-deficient diet increases DNA damage in mice myometrium.
Notes: (A) Immunohistochemical analyses of myometrial tissues of vitamin D deficiency and control groups by DNA damage marker γH2AX and DNA repair genes RAD50
and RAD51. All images were captured at 63× magnification, scale bar 200 µm, yellow arrows indicate YH2AX, green arrows indicate RAD50. (B) The degrees of DNA
damage and DNA repair were scored by counting the γH2AX, RAD50 and RAD51 positive nuclei. The percentage of positive cells was calculated based on the number of
stained cells, counted in a total of five random high-power fields in the representative myometrium of control and vitamin D3-deficiency samples. The average number of
cells is presented (n = 10, **P , 0.005 and ***P , 0.0005).
Abbreviation: Vit, vitamin.
of the tumoral microenvironment initiating transformation
from normal myometrium tissue to malignancies. This can
promote genomic instability characterized by defects in DNA
repair and accumulation of DNA damage.30,31 To test whether
vitamin D deficiency initiates DNA damage in myometrial
tissues, we performed immunohistochemical analyses
using immunoreactive γH2AX (a DNA damage marker) via
International Journal of Women’s Health 2018:10
polyclonal anti-γH2AX antibodies (Figure 6A). Vitamin Ddeficient mice demonstrated a remarkable increase in
γH2AX in myometrium tissues compared to healthy controls
(P , 0.05; Figure 6B). Conversely, vitamin D deficiency
induced a significant decrease in the expression of DNA
repair genes such as RAD50 (P , 0.005) and RAD51
(P ,0.005) compared to healthy controls (Figure 6B).
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elhusseini et al
Discussion
Our study provides a new model to explore the role of
vitamin D in the homeostasis of myometrium and associated
diseases such as UFs. The relationship between vitamin D
deficiency and increased risk of UF was confirmed in several
studies including our published data.4,32,33 A significant inverse
relationship between vitamin D serum levels and the severity
of fibroids was reported in African-American females, with a
higher disease burden detected in patients with low vitamin D
levels.4 The involvement of vitamin D in myometrial biology
was not examined in vivo previously. Our model of vitamin
D lacking function using 8-week exposure of C57/BL6 mice
to vitamin D-deficient diet provides evidence of different
functions accomplished by vitamin D in the murine uterus.
To date, numerous biologic functions have been reported for
vitamin D, including control of proliferation, inflammation,
autoimmunity, DNA damage/repair and tumorigenesis. Our
results demonstrate that vitamin D diet supplementation generates 80% of circulating vitamin D in the blood stream of the
mice and the remaining 20% are generated from other sources.
In addition, the present study establishes vitamin D as a key
molecule controlling sex steroid receptors, myometrium cell
proliferation, cytokine release and inflammation and DNA
repair in damaged cells in the murine myometrium.
Vitamin D exerts anti-inflammatory action by controlling
TGF-β and IL-10 to prevent immunosuppression and protect
the myometrium from transformation and tumorigenesis. Thus,
vitamin D deficiency promotes inflammation in myometrium
milieu and promotes the conversion and expansion of Tregs
from naïve CD4 T cells (a signature of tumor microenvironment). This initiates or, more likely, promotes transformation
of normal myometrium tissue to neoplasia after a mutagenic
event takes place, such as fibroid-causing Med12 mutations
in human fibroids.34 Indeed, previous studies have shown
that vitamin D induces autophagic cell death of cancer cells,
indicating the potential for vitamin D as an anti-fibroid
treatment.35,36 In the present study, we demonstrate that hypovitaminosis D induces the expression of progesterone receptors PRA and PRB and ER-α. Our data confirm our previous
observation of an inverse correlation between the upregulated
ER-α, PR-A and PR-B and expression of VDR in UFs.37
We also showed that treatment with vitamin D significantly
decreased levels of ER-α, PRA and PRB.17 Moreover, Wu
et al reported that PR-A was overexpressed in fibroid tissue
of African-American females compared to those of other
ethnic groups. This is consistent with the higher prevalence
of hypovitaminosis D in African-American women.2 The
current data show that vitamin D regulates cell survival and
512
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death by controlling PCNA, PARP and caspase-3. Control
of growth rate of the myometrium by vitamin D prevents the
development of fibrosis and establishment of chronic inflammation. Our in vivo model also confirms our prior in vitro
observation that vitamin D exhibits antifibrogenic effects
and modulates the profibrotic mediator TGFβR2.17 To date,
numerous studies showed the relationship of VDR and DNA
repair. It is established that vitamin D treatment stabilizes
VDR, BRCA1 and 53BP1 levels, preventing the recruitment
of these DNA repair factors to sites of damage in senescent
cells.38 Our present data demonstrate that lack of vitamin D
in vivo induces the phosphorylation of γH2AX, indicating
accumulation of DNA damage and genomic instability while
significantly decreasing RAD50 and RAD51 as markers of
DNA repair genes. These results suggest that vitamin D3 deficiency exacerbates DNA damage in myometrium and activate
UF cell growth through the reduction of DNA repair capacity.
Association of DNA damage and uncontrolled proliferation, in
the presence of chronic inflammation, will create a tumorigenic
niche. To date, it is established that tumor escape from immune
response is due to the immunosuppressive milieu.39,40 Studies
have shown that the tumor produces a high amount of suppressive cytokines (eg, TGF-β, IL-10), responsible for Tregs
conversion and proliferation, which suppresses the tumor
immune response.41–43 However, vitamin D supplementation
in vitamin D-deficient women with reproductive disorder
significantly decreases the bioavailability of TGF-β1.17
Conclusion
Our model validates our hypothesis that vitamin D deficiency
can work as a pro-tumorigenic promoting factor by starting
the first hit to render the myometrial tissue more vulnerable
to leiomyoma development, likely after the emergence of a
driver mutation such as Med12. The current results extend
our previous work on the pivotal role of vitamin D deficiency
in the pathogenesis of UF and establish the existence of an
interconnection between inflammation and DNA damage
promoting myometrial proliferation.
Acknowledgment
This study was supported by grant NIH-R01.
Disclosure
The authors report no conflicts of interest in this work.
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elhusseini et al
Supplementary material
Table S1 Sources for gene sequences and details of primer sequences used to amplify genes
Gene
name
Oligonucleotide primer sequences (5′ to 3′)
PCR
product size
Annealing
temperature (°C)
Number of
PCR cycles
TgF-β
F: CTTCGACGTGACAGACGCT;
r: gcaggggcagTgTaaacTTaTT
F: GCTGGACAACATACTGCTAACC;
r: aTTTccgaTaaggcTTggcaa
F: CGTCATTGCACGAAGACACAA;
r: ccTggTccaccaTTTTaaggc
210
60
40
190
60
40
220
60
40
Il-10
β-actin
Abbreviations: PCR, polymerase chain reaction; F, forward; R, reverse.
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