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Abnormal expression of the vitamin D receptor in keloid scars
burns 43 (2017) 1506 –1515
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Abnormal expression of the vitamin D receptor in keloid scars Jennifer M. Hahn a , Dorothy M. Supp a,b, * a
Research Department, Shriners Hospitals for Children—Cincinnati, Cincinnati,OH, USA Division of Plastic, Reconstructive and Hand Surgery/Burn Surgery, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA b
article info
abstract
Article history:
Keloids are abnormal fibroproliferative scars that pose a significant challenge to patients and
Accepted 6 April 2017
clinicians. The molecular basis for keloid formation remains incompletely understood, and currently no universally effective treatments exist. It is well recognized that keloids are more prevalent in populations with darkly pigmented skin, such as African Americans, but the basis for the link between skin color and keloid risk is not known. Pigmentation reduces
Keywords:
vitamin D production in the skin. Because most of the body’s vitamin D is produced in the
Keloid scar
skin, rates of vitamin D deficiency are higher in populations with darker skin pigmentation.
Nuclear hormone receptor
In addition to regulation of calcium homeostasis, vitamin D plays important roles in cell
Nuclear localization
proliferation, differentiation, cancer progression, inflammation, and fibrosis. The activities
Skin pigmentation
of vitamin D are dependent on the vitamin D receptor (VDR), a member of the steroid nuclear
Vitamin D
receptor superfamily. The ligand-bound VDR acts as a transcription factor; thus, nuclear
Vitamin D receptor
localization is required for ligand-dependent regulation of target gene expression. The current study investigated expression and nuclear localization of VDR in keloid scars (N=24) and biopsies of normal skin (N=24). Immunohistochemistry with two different antibodies demonstrated reduced VDR protein levels in a majority of keloid scars. Further, the percentage of epidermal cells displaying nuclear VDR localization was significantly lower in keloid scars compared with normal skin samples. Interestingly, analysis of VDR-positive nuclei among different normal skin samples showed a significant reduction in nuclear localization in epidermis of black donors compared with white donors. The results suggest that VDR may play a role in keloid pathology, and hint at a possible role for VDR in the increased susceptibility to keloid scarring in individuals with darkly pigmented skin. © 2017 Elsevier Ltd and ISBI. All rights reserved.
Abbreviations: ECM, extracellular matrix; EMT, epithelial mesenchymal transition; D3, 1,25-dihydroxyvitamin D3; TGF-b1, transforming growth factor-beta 1; VDR, vitamin D receptor; VDRE, vitamin D response element. * Corresponding author at: Shriners Hospitals for Children—Cincinnati, 3229 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail address: [email protected] (D.M. Supp). http://dx.doi.org/10.1016/j.burns.2017.04.009 0305-4179/© 2017 Elsevier Ltd and ISBI. All rights reserved.
burns 43 (2017) 1506 –1515
1.
Introduction
Advances in nearly all aspects of acute burn care in recent decades have significantly improved survival of burn patients. However, scarring continues to be a major problem for burn survivors that impacts quality of life and disrupts activities of daily living [1]. Keloid scars, in particular, are disfiguring and can cause pain, itching, decreased range of motion, and impaired psychosocial well-being [2–4]. Keloids are characterized by excessive and disorganized deposition of extracellular matrix (ECM) due to mechanisms that are incompletely understood. Keloids are significantly more common in darker skinned patients. For example, the estimated incidence of keloids is 1/30 among African Americans, approximately 20times the rate observed in the overall population [5]. It has been generally assumed that keloids do not occur in people with albinism, suggesting an important role for pigmentation in formation of keloid scars [6]. However, a recent epidemiological study of keloids in populations from two African countries identified people with albinism and keloid scarring, suggesting that genetic susceptibility may play an important role in keloid formation [7]. Despite evidence for a genetic predisposition for keloid scarring [5,7–9], no single gene has been identified, and the role of pigmentation in keloid formation remains poorly understood. Numerous signaling pathways have been implicated in the pathophysiology of keloid scarring, including the transforming growth factor-beta 1 (TGF-b1) pathway. TGF-b1, a pleiotropic cytokine involved in multiple aspects of wound repair, is a major regulator of fibrosis that stimulates proliferation and collagen production in keloid fibroblasts [10]. Additionally, TGF-b1 was found to regulate abnormal gene expression in keloid keratinocytes, contributing to a gene expression profile resembling epithelial mesenchymal transition (EMT) [11]. However, the signaling mechanisms upstream of TGF-b1 in keloid pathology have not been fully elucidated. A previous study suggested involvement of vitamin D in regulation of the response of keloid fibroblasts to TGF-b1 [12]. Treatment of keloid fibroblasts in vitro with the active form of vitamin D, 1,25-dihydroxyvitamin D3 (D3), reduced proliferation and inhibited the TGF-b1-induced expression of collagen [12]. Vitamin D plays important roles in calcium homeostasis, cell proliferation and differentiation, and is implicated in inhibition of cancer progression, inflammation, and fibrosis. Skin is the major source of vitamin D in humans, where epidermal keratinocytes synthesize the active form of vitamin D upon exposure to ultraviolet B (UVB) light in sunlight [13]. African Americans have a greater incidence of vitamin D insufficiency compared to non-Hispanic whites [14–16], which was proposed to result in part from reduced vitamin D production in darkly pigmented skin due to high levels of melanin, which shields keratinocytes from ultraviolet light [17–19]. Burn patients are at risk for vitamin D deficiency, which has been attributed to the inability to synthesize vitamin D in burned skin, metabolic alterations, lack of sun exposure during long hospitalizations, and inadequate supplementation after hospitalization [20–22]. Hypothetically, reduced vitamin D levels after burn may also contribute to the risk of abnormal scar formation, which may be further
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increased in certain patients due to pre-existing factors related to vitamin D deficiency, such as skin color or ethnic background. It was previously hypothesized that the incidence of abnormal scarring in African American populations may be linked to reduced production of vitamin D in darkly pigmented skin [23]; however, no such linkage has yet been described. The ability of vitamin D to regulate cellular processes is dependent on the vitamin D receptor (VDR), a member of the steroid nuclear receptor superfamily that is expressed in a wide variety of cells, including epidermal keratinocytes. Like other members of this nuclear receptor family, the ligandbound VDR acts as a transcription factor. When bound to D3, VDR binds to vitamin D response elements (VDREs) in the promoter regions of target genes to regulate their transcription [24,25]. Nuclear localization is required for ligand-dependent regulation of target gene transcription by VDR. In the absence of ligand, VDR is distributed in both the cytoplasm and nucleus; upon vitamin D treatment, cytoplasmic VDR translocates to the nucleus [26]. Expression of VDR is regulated, in part, by D3. One mechanism involves transcriptional regulation; when bound to D3, VDR regulates its own expression via VDREs in the gene’s enhancer region [24]. In addition, the VDR protein is stabilized by its ligand, extending the receptor’s halflife, which may in turn contribute to increased nuclear localization [24,27]. Expression of the VDR was previously identified in keloid fibroblasts, but has not been investigated in keloid epidermis. Expression of VDR was analyzed in uterine leiomyomas [28], which are benign fibrotic lesions with numerous similarities to keloids, including similarities in gene expression and high prevalence among African American women [29]. Western blot analysis demonstrated reduced VDR expression in over 60% of uterine fibroid tumors compared with adjacent normal myometrium [28]. Further, altered expression of VDR was observed in systemic sclerosis, a systemic connective tissue disorder that involves widespread fibrosis, including overproduction of collagen [30]. To determine if differences in VDR expression may be involved in keloid pathology, the current study investigated expression and nuclear localization of VDR in keloid scar tissue compared with normal skin.
2.
Materials and methods
2.1.
Collection of tissue samples
De-identified normal skin samples (N=24) were collected with University of Cincinnati (UC) Institutional Review Board (IRB) approval from breast or abdominal tissue from healthy donors undergoing elective plastic surgery procedures at the Shriners Hospitals for Children—Cincinnati and the UC Medical Center. Full thickness skin biopsies were collected after surgical excision from tissue designated as discard tissue. Because no protected health information (PHI) was collected from these donors, and only de-identified discard tissue was used, this was designated as “not human subjects research” by the UC IRB; therefore, patient consent was not required. For these deidentified samples, only patient age (in years), gender, and race/ethnicity were recorded. All skin samples were assigned a sequential identification number upon collection.
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Keloid scar patients were enrolled with informed consent in a study approved by the UC IRB. Patients were enrolled based on previously scheduled procedures for elective scar excision at the Shriners Hospitals for Children—Cincinnati, or the UC Medical Center. Biopsies of keloid scars were collected after scar excision from tissue designated as “discard” tissue. When possible (scars larger than 1cm2 in area), biopsies for histological sections were collected from the central region of each scar. The scars were identified as keloids based on clinical diagnosis by each patient’s physician and/or surgeon, which involved consideration of scar progression over time and expansion beyond the original wound boundary. Following keloid excision, diagnosis was confirmed by analysis of histological sections, including identification of keloidal collagen. Scars were excluded from the analysis if there was ambiguity in diagnosis. A total of 21 biopsies were collected from 19 distinct, unrelated donors. Two patients had multiple scars removed at different times after injury; these patients are indicated by asterisks in Supplemental Table 1. Patient demographic data collected for this study included date of birth, gender, and race/ethnicity for all patients. In addition, the date and mode of injury, the duration of the scar (e.g., time since the scar was first noticed or diagnosed as keloid), and any prior treatments, if known, were also recorded. In addition to keloid patients enrolled via informed consent, three additional keloid scars were collected as de-identified samples. For these scar samples, only the age, gender, and race/ethnicity of donors were recorded. Race/ethnicity classifications were based on patient selfidentification or, for younger patients, the guardian’s designation. Patients classified as “White” included non-Hispanic Whites/Caucasians. Patients classified as “Black” included those who self-identified as Black or African American, and included one patient who self-identified as “Black/mixed race.” Hispanic, Asian, and Native American patients were not included in the current study.
with an Eclipse 90i microscope and photographed with a DSRi1 Digital Microscope Camera (Nikon Instruments Inc., Melville, NY). Colorimetric immunostaining was used for quantitation of nuclei staining positive for VDR. Immunohistochemistry was performed using a different rabbit polyclonal anti-VDR antibody (Abcam; catalog #ab134826) that recognizes an internal sequence of the human vitamin D receptor. Detection utilized the Vectastain Elite ABC HRP Kit (Peroxidase, Universal; Vector Laboratories) followed by incubation (45s for all sections) with ImmPact NovaRed Peroxidase (HRP) Substrate (Vector Laboratories). Counterstaining of nuclei was performed by a brief (10s) incubation with Vector Hematoxylin QS (Vector Laboratories). Sections were all processed identically to minimize variability due to technical factors. For quantification of the percent of nuclei staining positive for VDR, three non-overlapping images per sample were photographed at 40X magnification (equivalent to 300 mm of epidermis). All nuclei were counted in each microscopic field, and all nuclei showing any level of positive staining for VDR were counted, enabling calculation of the percent of nuclei staining positive for VDR. Cells displaying any level of nuclear VDR-specific staining were counted as positive, even if there was little or no distinction between nuclear and cytoplasmic staining levels. For each tissue sample, two negative controls were performed on serial sections, omitting either the primary antibody or the biotinylated secondary antibody but including the hematoxylin nuclear stain. VDR-stained sections were carefully compared to negative control sections to distinguish between staining due to VDR immunolocalization and pigment due to epidermal melanin in some samples. For each field, the percent of nuclei staining positive for VDR (% VDR+) was determined, and a mean for each tissue sample was calculated.
2.2. Immunohistochemistry and quantification of nuclear localization
Statistical analyses were performed using SigmaPlot for Windows Version 11.0 (SyStat Software, Inc., San Jose, CA). Pairwise comparisons of numeric values were analyzed by ttest. Pairwise comparisons of categorical data were analyzed using the Chi-square test. Quantitative data are presented as meansstandard deviations. Differences were considered statistically significant at p<0.05.
Tissue biopsies were fixed in 10% buffered neutral formalin. Tissues were processed, including embedding in paraffin and sectioning, by the Histology Core Facility at Shriners Hospitals for Children—Cincinnati. Slides containing 4-mm thick paraffin sections were incubated for 2h at 55 C and deparaffinized by incubation in xylene followed by a graded alcohol series [31]. Antigen retrieval was performed using Citric Acid Based Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) and tissue was permeabilized in 0.05% Triton X-100 (Sigma–Aldrich, St. Louis, MO). For fluorescent labeling of VDR in tissue sections, a rabbit polyclonal anti-VDR antibody was used (Abcam, Cambridge, MA; catalog #ab3508). This antibody recognizes an epitope near the C-terminus of the human vitamin D receptor (amino acids 395-413). For fluorescent detection, Alexa Fluor 488conjugated Chicken Anti-rabbit IgG (H+L) was used as a secondary antibody (ThermoFisher Scientific, Waltham, MA; catalog #A-21441). Coverslips were mounted using 1 Vectashield Mounting Medium with DAPI (40 ,6-diamidino2-phenylindole; Vector Laboratories). Sections were viewed
2.3.
Statistical analyses
3.
Results
3.1.
Study population
Localization of VDR protein was analyzed in 24 normal skin samples and 24 keloid scar samples (Table 1). Normal skin donors ranged in age from 14 to 65 years (median age, 30.0 years); keloid scar patients ranged in age from 4 to 51 years (median 15.5 years). The mean age for normal skin donors was significantly older than the mean for keloid scar donors (31.5 13.3 years vs. 17.512.1 years, respectively; p<0.001). There was also a statistically significant difference in donor gender between the two groups, with one male and 23 females in the normal skin group, and 15 males and 9 females in the keloid
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Table 1 – Scar and skin samples used in study, and percent VDR-positive nuclei. Keloid scars: ID#
Ethnicity
Gender
Age (years)
Body site
%VDR+nuclei (meanstd. dev.)
753K 794K 795K 797K 798K 810K 817K 818K 829K 830K 835K 837K 850K 860K 868K 874K 878K 881K 884K 885K 897K 919K 934K 951K
W W B W W B W B B B B B W B B B B B B B Ba B B B
M M F M M M M M F M M F M M M M M M F F F F F F
11 17 20 10 17 13 8 16 12 17 17 4 16 9 13 22 17 37 51 10 8 10 15 51
Chin Chin Neck Ear Ear Arm Ear Shoulder Ear Neck Face Ear Face Face Chin Back Scapula Ear Neck Back Ear Ear Ear Breast
40.7 18.9 8.4 12.7 4.8 2.6 30.0 4.0 0.0 0.0 4.4 2.7 58.4 13.7 1.3 1.1 0.5 0.9 23.1 5.6 9.1 5.0 0.9 0.8 36.2 7.6 44.6 12.0 35.4 6.2 15.2 5.4 5.9 1.0 34.7 0.4 1.5 2.6 12.7 13.6 61.2 12.2 2.8 2.6 0.0 0.0 11.6 3.2
%VDR+nuclei (meanstd. dev.)
Normal skin ID#
Ethnicity
Gender
Age (years)
Body Site
822 824 831 844 858 859 861 863 865 866 869 870 876 880 882 886 894 896 898 916 920 925 927 929
W B W B W W W B B W B W B W W B W W B B B B B W
F F F F F F F F F F F F F F F M F F F F F F F F
42 24 38 38 42 38 40 55 52 30 24 30 18 15 65 17 14 26 25 25 20 30 31 17
Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Abdomen Breast Abdomen Breast Breast Breast Breast Breast Breast
a
51.7 41.7 75.5 42.9 67.5 74.2 74.0 47.7 74.6 61.4 76.5 47.3 47.3 67.8 50.7 53.4 65.2 85.1 39.8 21.6 29.5 63.6 31.0 64.9
4.2 2.0 9.8 9.8 8.2 12.8 6.8 10.0 13.0 10.5 5.2 16.1 2.0 2.1 13.5 6.8 5.5 1.9 5.9 7.6 8.8 20.2 7.0 7.3
Abbreviations: W, white; B, Black or African American; F, female; M, male. Patient (897K) self-identified as African American and mixed race, but had light skin.
group (p<0.001). The normal skin group contained an equal number of black and white donors, whereas the keloid group had more black than white donors; however, the difference in racial makeup between the two groups was not statistically significant (p=0.136).
Normal skin samples were obtained primarily from breast tissue (22/24), with two samples obtained from abdominal tissue. In contrast, keloid scars were from a variety of body sites, including ear, face, chin, neck, back, scapula/shoulder, breast, and arm (Table 1). The mode of initial injury was known
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Fig. 1 – VDR localization in keloid scar and normal skin epidermis. VDR localization is shown in normal skin (A–I) and in keloid scars (J–R). Representative sections are shown. Fluorescent detection of VDR (green) is shown in the left column (A, D, G, J, M, P), nuclear staining with DAPI (blue) is shown in the center column (B, E, H, K, N, Q), and merged images are shown in the right
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Fig. 2 – Variable VDR staining and nuclear localization in normal skin and keloid scar epidermis from black and white donors. Shown are sections of normal skin (A–H) and keloid scar (I–P) from black (A–D, I–L) and white (E–H, M–P) donors. Donor identification numbers are indicated in each panel. For each pair of images, the first (A, C, E, G, I, K, M, O) shows colorimetric localization of VDR, and the second (B, D, F, H, J, K, N, P) shows a negative control section from the same donor processed without addition of primary anti-VDR antibody. Insets (G, O) show higher magnification images of boxed areas in these sections; examples of VDR-positive nuclei are indicated by arrowheads, and VDR-negative nuclei are indicated by arrows. The scale bar in A is the same for all panels: 50 mm.
for 19 of the 24 keloids (Supplemental Table 1). The majority (14/19) resulted from burns, and one keloid formed in an autograft donor site in a burn patient. Of the eight ear keloids with known etiologies, four occurred following ear piercing, and four were the result of burn.
3.2.
Expression and nuclear localization of VDR
Immunohistochemistry was initially performed using a fluorescent detection method. Comparisons among different samples from both keloid scar tissue and normal skin showed
variable levels of expression, both in dermal and epidermal VDR staining levels. Variability in overall expression levels was observed between groups, and among different donors within the same group. However, a striking difference was observed between normal skin and keloid scar epidermis: reduced nuclear localization of VDR protein was observed in keloid scar keratinocytes compared with normal skin (Fig. 1). In normal skin, bright staining of nuclei was clearly evident, with much lower levels of cytoplasmic staining observed (Fig. 1A–I). In contrast, staining for VDR in keloid scar epidermis was more diffuse, and although some nuclear localization was observed,
column (C, F, I, L, O, R). Insets (G–I and P–R) show higher magnification images of boxed areas in these sections; examples of VDRpositive nuclei are indicated by arrowheads, and VDR-negative nuclei are indicated by arrows. The scale bar in A is the same for all panels: 50 mm.
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this was patchy and levels were greatly reduced comparedwith normal skin (Fig. 1J–R). Expression levels and localization patterns varied among different keloid scar samples. Some had diffuse cytoplasmic staining with little distinction of staining levels between cytoplasm and nucleus, or with occasional nuclei showing increased VDR staining (Fig. 1J–O). In other keloids, epidermal VDR staining was mainly observed in the cytoplasm, with few nuclei positive for VDR signal (Fig. 1P–R). Positive VDR staining was observed in the dermal regions of both normal skin and keloid scar; expression levels were variable and no consistent differences between groups were observed. Collagen autofluorescence was observed in some samples, particularly in normal skin, complicating comparisons between groups using this staining technique. To confirm the specificity of the observed epidermal staining patterns, immunohistochemistry was performed using a second anti-VDR antibody directed at a different region of the VDR protein. Colorimetric immunostaining was performed using identical staining parameters for all samples, to enable comparisons of both localization and staining levels. Although variable staining levels were observed between samples from different individuals within each group, overall VDR expression levels were higher in normal skin compared with keloid scar epidermis (Fig. 2). Further, differences in nuclear localization of VDR were observed between normal skin and keloid scar epidermis. Nuclear localization was observed in all 24 normal skin
Fig. 3 – Quantitative analysis of VDR nuclear localization. The percent of epidermal nuclei staining positive for VDR (mean +standard deviation) is shown for normal skin and keloid scar samples (N=24 per group). The difference between groups was statistically significant (p<0.001).
samples, whereas some keloid scars showed little to no nuclear localization (Fig. 2). To quantify differences in VDR nuclear localization, the percent of epidermal nuclei showing positive localization of VDR was calculated for each sample (Table 1). For this quantitative analysis, only nuclear localization was considered; any level of VDR-specific staining in the nucleus was considered a positive signal. The percent of nuclei staining positive for VDR was variable in keloid scars, and no correlation with age, gender, body site, or area of keloid scar was found (Table 1, Supplemental Table 1 and data not shown). A comparison of all normal and keloid scar samples (N=24 each) showed that the percent of epidermal nuclei staining positive for VDR was significantly higher in normal skin compared with keloid scar (Fig. 3). To determine if skin color is associated with nuclear localization, analysis was performed on sub-groups based on the self-identified racial/ ethnic groups of donors. This analysis showed that normal epidermis of white donors displayed significantly higher levels of VDR nuclear localization compared with keloid scars from white donors; a significant difference was also observed between normal skin and keloid scar from black donors (Fig. 4). Interestingly, comparison of normal epidermis from black and white donors (N=12 each) revealed a significant difference in VDR nuclear localization, with higher levels in skin of white donors (Fig. 4). In keloid scars, no significant difference in nuclear localization was found between black (N=18) and white (N=8) donors; however, this analysis was not sufficiently powered to detect a possible significant difference due to the relatively low number of keloid scars from white donors.
Fig. 4 – Quantitative analysis of VDR nuclear localization in normal skin and keloid scar according to skin color. The percent of epidermal nuclei staining positive for VDR (mean +standard deviation) is shown for normal skin of white (N–W) and black (N–B) donors, and for keloid scars of white (K–W) and black (K–B) donors. Statistically significant differences are indicated (*p<0.001; # p<0.01).
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4.
Discussion
The results demonstrate significantly lower nuclear localization of VDR in keloid scar epidermis compared with normal skin. As a member of the nuclear hormone receptor superfamily, VDR activity involves transcriptional regulation of target genes. Therefore, reduced levels of nuclear localization may result in changes in the expression of downstream target genes. In systemic sclerosis, which is characterized by excessive deposition of ECM, VDR expression was reduced, and VDR knockdown was shown to enhance the fibrotic response of fibroblasts to TGF-b1 [30]. VDR expression was also reported to be reduced in many uterine fibroids, and treatment of uterine fibroid cells in vitro with D3 induced VDR expression and reduced expression of ECM genes [28]. Antifibrotic activity of D3 has been demonstrated in other organ systems and cell types, including lung, kidney, and liver [32–34]. D3 has been implicated in regulation of genes involved in EMT [35], a process implicated in fibrosis and cancer metastasis, as well as keloid scarring [11]. Knockdown or overexpression of VDR in a breast cancer cell line resulted in either increased or decreased expression of EMT markers, respectively [36]. Further, D3 acting via the VDR has been shown to exhibit immunomodulatory and anti-inflammatory properties in numerous cell types and disease models [37,38]. These previous studies suggest potential mechanisms whereby reduced VDR expression and nuclear localization may contribute to keloid pathology, by enhancing the responsiveness of cells to TGFb1 signaling and favoring EMT-like cellular changes and/or by modulating inflammation. However, increased TGFb1 expression in keloids may contribute to abnormal expression of VDR. Previous studies showed that persistent exposure of normal human dermal fibroblasts to TGF-b1 reduced VDR expression levels in vitro, suggesting complex crosstalk between these signaling pathways [30]. Further studies will be needed to determine the mechanism(s) whereby reduced VDR expression contributes to keloid pathology. In addition to differences in nuclear localization between keloid scars and normal skin, a significant difference was observed between normal skin samples from white and black donors. To our knowledge, this has not been previously reported. Although this result should be confirmed in a larger population, it suggests a possible mechanism for increased risk of keloid formation in skin of color. The basis for differential VDR localization in light vs. dark skin is unclear, but may be related to differences in vitamin D levels. African Americans have a greater incidence of vitamin D insufficiency compared to non-Hispanic whites [14,16,39,40]. Insufficiency results primarily from reduced vitamin D production in pigmented skin due to high levels of melanin, which shields keratinocytes from UVB [17]. Any alteration in the level of UVB reaching the skin impacts vitamin D production. In addition, diet plays an important role in vitamin D levels, and factors such as inadequate nutrition, cultural preferences, and lack of supplementation can influence vitamin D deficiency [16,41,42]. Vitamin D is known to regulate expression of VDR via VRDEs in the enhancer region of the VDR gene [24]. Hypothetically, vitamin D deficiency may contribute to reduced VDR expression or nuclear localization. Increased
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nuclear localization of VDR has been observed in response to D3 binding in vitro [43,44]. Previous studies demonstrated a two-fold increase in VDR protein levels in nuclear extracts from human skin treated topically with vitamin D in vivo [27]. This was due to reduced degradation upon ligand binding, which allowed VDR levels to increase in the nucleus [27]. This indicates that cells displaying reduced nuclear localization of VDR are still capable of responding to vitamin D by increasing VDR nuclear translocation and, hypothetically, increasing transcription of VDR target genes. This is relevant for the potential use of vitamin D as a therapeutic agent for abnormal scarring because it suggests the feasibility of modulating vitamin D-regulated gene expression in individuals with reduced basal nuclear VDR levels. This study had several limitations that resulted from our study design, which involved analysis of normal skin and keloid scar samples obtained from different donors. Because of this, the normal skin and keloid scar groups differed in several features including gender, age, and genetic background. Collection of a non-lesional skin biopsy from an individual predisposed to keloid scarring is likely to result in new keloid scar formation. This was of particular concern for the current study because the majority of keloid scar samples were collected from pediatric patients. Therefore, due to ethical considerations, we did not collect non-lesional skin biopsies from keloid patients for the purpose of this study. Whether samples were collected from de-identified tissue or from patients enrolled by informed consent, tissue was only used if it would otherwise be discarded. There were significant differences in age and gender between normal skin and keloid scar donors. The difference in age is due in part to the patient population, as samples were collected from patients at a pediatric burn specialty hospital as well as a medical center that treats predominantly adult patients. Normal skin samples were collected from elective breast reductions and abdominoplasties; the majority of these patients (19/24) were adults (>18 years of age). Most of the keloid scars were obtained from patients initially treated at a pediatric burn hospital, therefore the majority of these patients (19/24) were age 18 years or younger. However, the younger age range for keloid scars is also related to the prevalence of keloids in younger patients; keloid scars are most commonly observed in patients between the ages of 15 and 24 years [45]. Because the majority of plastic surgery procedures resulting in normal discard skin from healthy donors are performed in women, the majority of normal skin samples were from breast tissue collected from females. Although there were more male than female keloid patients, the distribution was not significantly different than a 50:50 distribution of males and females (data not shown). In contrast to the normal skin samples, keloid scars were from a variety of body sites. Although ears are among the most common sites for keloid formation [45], only nine of the keloids in the current study were ear keloids. Differences in body location may be relevant to VDR expression because VDR expression in skin is induced by UVB [46]. In the current study, the normal skin samples were from breast and abdominal skin, which are areas likely to be protected from UVB exposure. Thus, if body site influenced VDR expression due to sun exposure, the normal skin samples might be
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expected to show reduced expression compared with keloid samples from areas with greater potential for sun exposure. Vitamin D deficiency is common following major burn, and has been attributed to the inability to efficiently synthesize vitamin D in burned skin, metabolic alterations, lack of sun exposure during long hospitalizations, and inadequate supplementation [21,22,47]. A recent study found a correlation between vitamin D levels a year or more after extensive burn and scar formation [48]. Vitamin D levels were lower in patients with higher scores on the Modified Vancouver Scar Scale (MVSS), which indicates more severe scarring [48]. The authors speculated that patients with burn scars may have avoided sun exposure, leading to vitamin D deficiency [48]. This led them to recommend screening of patients with high MVSS scores for vitamin D deficiency. It would be interesting in future studies to determine whether vitamin D deficiency is predictive of abnormal scar formation.
Conflicts of interest The authors report no conflicts of interest.
Acknowledgments This work was supported by the Shriners Hospitals for Children (Medical Research Grant #85300). The authors thank the patients of the Shriners Hospital for Children—Cincinnati and the University of Cincinnati Medical Center who generously donated tissue samples for this study, and the surgeons and research nurses who assisted in collection of these samples. The authors also thank Kevin McFarland and Kelly Combs for critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. burns.2017.04.009. REFERENCES
[1] Stella M, Castagnoli C, Gangemi EN. Postburn scars: an update. Int J Low Extremity Wounds 2008;7:176–81. [2] Bock O, Schmid-Ott G, Malewski P, Mrowietz U. Quality of life of patients with keloid and hypertrophic scarring. Arch Dermatol Res 2006;297:433–8. [3] Van Loey NE, Van Son MJ. Psychopathology and psychological problems in patients with burn scars: epidemiology and management. Am J Clin Dermatol 2003;4:245–72. [4] Balci DD, Inandi T, Dogramaci CA, Celik E. DLQI scores in patients with keloids and hypertrophic scars: a prospective case control study. J Dtsch Dermatol Ges 2009;7:688–92. [5] Velez Edwards DR, Tsosie KS, Williams SM, Edwards TL, Russell SB. Admixture mapping identifies a locus at 15q21: 2– 22.3 associated with keloid formation in African Americans. Hum Genet 2014;133:1513–23. Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG. Hypertrophic scarring and keloids: pathomechanisms and
[6] current and emerging treatment strategies. Mol Med 2011;17:113–25. [7] Kiprono SK, Chaula BM, Masenga JE, Muchunu JW, Mavura DR, Moehrle M. Epidemiology of keloids in normally pigmented Africans and African people with albinism: population-based cross-sectional survey. Br J Dermatol 2015;173:852–4. [8] Shih B, Bayat A. Genetics of keloid scarring. Arch Dermatol Res 2010;302:319–39. [9] Brown JJ, Bayat A. Genetic susceptibility to raised dermal scarring. Br J Dermatol 2009;161:8–18. [10] Andrews JP, Marttala J, Macarak E, Rosenbloom J, Uitto J. Keloids: the paradigm of skin fibrosis—pathomechanisms and treatment. Matrix Biol 2016;51:37–46. [11] Hahn JM, McFarland KL, Combs KA, Supp DM. Partial epithelial-mesenchymal transition in keloid scars: regulation of keloid keratinocyte gene expression by transforming growth factor-ß1. Burns Trauma 2016;4:30. [12] Zhang GY, Cheng T, Luan Q, Liao T, Ni CL, Zheng X, et al. Vitamin D: a novel therapeutic approach for keloid, an in vitro analysis. Br J Dermatol 2011;164:729–37. [13] Bikle DD. Vitamin D and the skin. J Bone Miner Metab 2010;28:117–30. [14] Ginde AA, Liu MC, Camargo Jr. CA. Demographic differences and trends of vitamin D insufficiency in the US population, 1988–2004. Arch Intern Med 2009;169:626–32. [15] Engelman CD, Fingerlin TE, Langefeld CD, Hicks PJ, Rich SS, Wagenknecht LE, et al. Genetic and environmental determinants of 25-hydroxyvitamin D and 1,25dihydroxyvitamin D levels in Hispanic and African Americans. J Clin Endocrinol Metab 2008;93:3381–8. [16] Harris SS. Vitamin D and African Americans. J Nutr 2006;136:1126–9. [17] Holick MF. Vitamin D: a millenium perspective. J Cell Biochem 2003;88:296–307. [18] Armas LA, Dowell S, Akhter M, Duthuluru S, Huerter C, Hollis BW, et al. Ultraviolet-B radiation increases serum 25hydroxyvitamin D levels: the effect of UVB dose and skin color. J Am Acad Dermatol 2007;57:588–93. [19] Libon F, Cavalier E, Nikkels AF. Skin color is relevant to vitamin D synthesis. Dermatology 2013;227:250–4. [20] Wray CJ, Mayes T, Khoury J, Warden GD, Gottschlich M. The 2002 Moyer Award. Metabolic effects of vitamin D on serum calcium, magnesium, and phosphorus in pediatric burn patients. J Burn Care Rehabil 2002;23:416–23. [21] Gottschlich MM, Mayes T, Khoury J, Warden GD. Hypovitaminosis D in acutely injured pediatric burn patients. J Am Diet Assoc 2004;104:931–41 quiz. [22] Klein GL, Herndon DN, Chen TC, Kulp G, Holick MF. Standard multivitamin supplementation does not improve vitamin D insufficiency after burns. J Bone Miner Metab 2009;27:502–6. [23] Cooke GL, Chien A, Brodsky A, Lee RC. Incidence of hypertrophic scars among African Americans linked to vitamin D-3 metabolism? J Natl Med Assoc 2005;97:1004–9. [24] Saccone D, Asani F, Bornman L. Regulation of the vitamin D receptor gene by environment, genetics and epigenetics. Gene 2015;561:171–80. [25] Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ. The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer 2014;14:342–57. [26] Racz A, Barsony J. Hormone-dependent translocation of vitamin D receptors is linked to transactivation. J Biol Chem 1999;274:19352–60. [27] Li XY, Boudjelal M, Xiao JH, Peng ZH, Asuru A, Kang S, et al. 1,25-Dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin. Mol Endocrinol 1999;13:1686–94. [28] Halder SK, Osteen KG, Al-Hendy A. 1,25-Dihydroxyvitamin d3 reduces extracellular matrix-associated protein expression in human uterine fibroid cells. Biol Reprod 2013;89:150.
burns 43 (2017) 1506 –1515
[29] Luo X, Pan Q, Liu L, Chegini N. Genomic and proteomic profiling II: comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars. Reprod Biol Endocrinol 2007;5:35. [30] Zerr P, Vollath S, Palumbo-Zerr K, Tomcik M, Huang J, Distler A, et al. Vitamin D receptor regulates TGF-beta signalling in systemic sclerosis. Ann Rheum Dis 2014;74:e20. [31] Osborn M, Isenberg S. Immunocytochemistry of frozen and of paraffin tissue sections. In: Celis JE, editor. Cell biology: a laboratory handbook. San Diego, CA: Academic Press; 1998. p. 486–92. [32] Ramirez AM, Wongtrakool C, Welch T, Steinmeyer A, Zugel U, Roman J. Vitamin D inhibition of pro-fibrotic effects of transforming growth factor beta1 in lung fibroblasts and epithelial cells. J Steroid Biochem Mol Biol 2010;118:142–50. [33] Zhang Y, Kong J, Deb DK, Chang A, Li YC. Vitamin D receptor attenuates renal fibrosis by suppressing the renin-angiotensin system. J Am Soc Nephrol 2010;21:966–73. [34] Abramovitch S, Han-Bachar L, Sharvit E, Weisman Y, Ben TA, Brazowski E, et al. Vitamin D inhibits proliferation and profibrotic marker expression in hepatic stellate cells and decreases thioacetamide-induced liver fibrosis in rats. Gut 2011;60:1728–37. [35] Larriba MJ, Garcia de HA, Munoz A. Vitamin D and the epithelial to mesenchymal transition. Stem Cells Int 20162016: 6213872. [36] Pervin S, Hewison M, Braga M, Tran L, Chun R, Karam A, et al. Down-regulation of vitamin D receptor in mammospheres: implications for vitamin D resistance in breast cancer and potential for combination therapy. PLoS One 2013;8:e53287. [37] Lin Z, Li W. The roles of vitamin D and its analogs in inflammatory diseases. Curr Top Med Chem 2016;16:1242–61. [38] Vojinovic J. Vitamin D receptor agonists’ anti-inflammatory properties. Ann N Y Acad Sci 2014;1317:47–56.
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[39] Weishaar T, Vergili JM. Vitamin D status is a biological determinant of health disparities. J Acad Nutr Diet 2013;113:643–51. [40] Au LE, Harris SS, Dwyer JT, Jacques PF, Sacheck JM. Association of serum 25-hydroxyvitamin D with race/ethnicity and constitutive skin color in urban schoolchildren. J Pediatr Endocrinol Metab 2014;27:1095–100. [41] Macdonald HM. Contributions of sunlight and diet to vitamin D status. Calcif Tissue Int 2013;92:163–76. [42] Bailey RK, Fileti CP, Keith J, Tropez-Sims S, Price W, LisonOttey SD. Lactose intolerance and health disparities among African Americans and Hispanic Americans: an updated consensus statement. J Natl Med Assoc 2013;105:112–27. [43] Walters SN, Reinhardt TA, Dominick MA, Horst RL, Littledike ET. Intracellular location of unoccupied 1,25dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium. Arch Biochem Biophys 1986;246:366–73. [44] Yasmin R, Williams RM, Xu M, Noy N. Nuclear import of the retinoid X receptor, the vitamin D receptor, and their mutual heterodimer. J Biol Chem 2005;280:40152–60. [45] Trace AP, Enos CW, Mantel A, Harvey VM. Keloids and hypertrophic scars: a spectrum of clinical challenges. Am J Clin Dermatol 2016;17:201–23. [46] Demetriou SK, Ona-Vu K, Teichert AE, Cleaver JE, Bikle DD, Oh DH. Vitamin D receptor mediates DNA repair and is UV inducible in intact epidermis but not in cultured keratinocytes. J Invest Dermatol 2012;132:2097–100. [47] Klein GL, Chen TC, Holick MF, Langman CB, Price H, Celis MM, et al. Synthesis of vitamin D in skin after burns. Lancet 2004;363:291–2. [48] Terzi R, Guven M. Bone mineral density after burn injury and its relation to the characteristics of scar tissue. J Burn Care Res 2016;37:e263–7.
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Abnormal expression of the vitamin D receptor in keloid scars Jennifer M. Hahn a , Dorothy M. Supp a,b, * a
Research Department, Shriners Hospitals for Children—Cincinnati, Cincinnati,OH, USA Division of Plastic, Reconstructive and Hand Surgery/Burn Surgery, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA b
article info
abstract
Article history:
Keloids are abnormal fibroproliferative scars that pose a significant challenge to patients and
Accepted 6 April 2017
clinicians. The molecular basis for keloid formation remains incompletely understood, and currently no universally effective treatments exist. It is well recognized that keloids are more prevalent in populations with darkly pigmented skin, such as African Americans, but the basis for the link between skin color and keloid risk is not known. Pigmentation reduces
Keywords:
vitamin D production in the skin. Because most of the body’s vitamin D is produced in the
Keloid scar
skin, rates of vitamin D deficiency are higher in populations with darker skin pigmentation.
Nuclear hormone receptor
In addition to regulation of calcium homeostasis, vitamin D plays important roles in cell
Nuclear localization
proliferation, differentiation, cancer progression, inflammation, and fibrosis. The activities
Skin pigmentation
of vitamin D are dependent on the vitamin D receptor (VDR), a member of the steroid nuclear
Vitamin D
receptor superfamily. The ligand-bound VDR acts as a transcription factor; thus, nuclear
Vitamin D receptor
localization is required for ligand-dependent regulation of target gene expression. The current study investigated expression and nuclear localization of VDR in keloid scars (N=24) and biopsies of normal skin (N=24). Immunohistochemistry with two different antibodies demonstrated reduced VDR protein levels in a majority of keloid scars. Further, the percentage of epidermal cells displaying nuclear VDR localization was significantly lower in keloid scars compared with normal skin samples. Interestingly, analysis of VDR-positive nuclei among different normal skin samples showed a significant reduction in nuclear localization in epidermis of black donors compared with white donors. The results suggest that VDR may play a role in keloid pathology, and hint at a possible role for VDR in the increased susceptibility to keloid scarring in individuals with darkly pigmented skin. © 2017 Elsevier Ltd and ISBI. All rights reserved.
Abbreviations: ECM, extracellular matrix; EMT, epithelial mesenchymal transition; D3, 1,25-dihydroxyvitamin D3; TGF-b1, transforming growth factor-beta 1; VDR, vitamin D receptor; VDRE, vitamin D response element. * Corresponding author at: Shriners Hospitals for Children—Cincinnati, 3229 Burnet Avenue, Cincinnati, OH 45229, USA. E-mail address: [email protected] (D.M. Supp). http://dx.doi.org/10.1016/j.burns.2017.04.009 0305-4179/© 2017 Elsevier Ltd and ISBI. All rights reserved.
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1.
Introduction
Advances in nearly all aspects of acute burn care in recent decades have significantly improved survival of burn patients. However, scarring continues to be a major problem for burn survivors that impacts quality of life and disrupts activities of daily living [1]. Keloid scars, in particular, are disfiguring and can cause pain, itching, decreased range of motion, and impaired psychosocial well-being [2–4]. Keloids are characterized by excessive and disorganized deposition of extracellular matrix (ECM) due to mechanisms that are incompletely understood. Keloids are significantly more common in darker skinned patients. For example, the estimated incidence of keloids is 1/30 among African Americans, approximately 20times the rate observed in the overall population [5]. It has been generally assumed that keloids do not occur in people with albinism, suggesting an important role for pigmentation in formation of keloid scars [6]. However, a recent epidemiological study of keloids in populations from two African countries identified people with albinism and keloid scarring, suggesting that genetic susceptibility may play an important role in keloid formation [7]. Despite evidence for a genetic predisposition for keloid scarring [5,7–9], no single gene has been identified, and the role of pigmentation in keloid formation remains poorly understood. Numerous signaling pathways have been implicated in the pathophysiology of keloid scarring, including the transforming growth factor-beta 1 (TGF-b1) pathway. TGF-b1, a pleiotropic cytokine involved in multiple aspects of wound repair, is a major regulator of fibrosis that stimulates proliferation and collagen production in keloid fibroblasts [10]. Additionally, TGF-b1 was found to regulate abnormal gene expression in keloid keratinocytes, contributing to a gene expression profile resembling epithelial mesenchymal transition (EMT) [11]. However, the signaling mechanisms upstream of TGF-b1 in keloid pathology have not been fully elucidated. A previous study suggested involvement of vitamin D in regulation of the response of keloid fibroblasts to TGF-b1 [12]. Treatment of keloid fibroblasts in vitro with the active form of vitamin D, 1,25-dihydroxyvitamin D3 (D3), reduced proliferation and inhibited the TGF-b1-induced expression of collagen [12]. Vitamin D plays important roles in calcium homeostasis, cell proliferation and differentiation, and is implicated in inhibition of cancer progression, inflammation, and fibrosis. Skin is the major source of vitamin D in humans, where epidermal keratinocytes synthesize the active form of vitamin D upon exposure to ultraviolet B (UVB) light in sunlight [13]. African Americans have a greater incidence of vitamin D insufficiency compared to non-Hispanic whites [14–16], which was proposed to result in part from reduced vitamin D production in darkly pigmented skin due to high levels of melanin, which shields keratinocytes from ultraviolet light [17–19]. Burn patients are at risk for vitamin D deficiency, which has been attributed to the inability to synthesize vitamin D in burned skin, metabolic alterations, lack of sun exposure during long hospitalizations, and inadequate supplementation after hospitalization [20–22]. Hypothetically, reduced vitamin D levels after burn may also contribute to the risk of abnormal scar formation, which may be further
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increased in certain patients due to pre-existing factors related to vitamin D deficiency, such as skin color or ethnic background. It was previously hypothesized that the incidence of abnormal scarring in African American populations may be linked to reduced production of vitamin D in darkly pigmented skin [23]; however, no such linkage has yet been described. The ability of vitamin D to regulate cellular processes is dependent on the vitamin D receptor (VDR), a member of the steroid nuclear receptor superfamily that is expressed in a wide variety of cells, including epidermal keratinocytes. Like other members of this nuclear receptor family, the ligandbound VDR acts as a transcription factor. When bound to D3, VDR binds to vitamin D response elements (VDREs) in the promoter regions of target genes to regulate their transcription [24,25]. Nuclear localization is required for ligand-dependent regulation of target gene transcription by VDR. In the absence of ligand, VDR is distributed in both the cytoplasm and nucleus; upon vitamin D treatment, cytoplasmic VDR translocates to the nucleus [26]. Expression of VDR is regulated, in part, by D3. One mechanism involves transcriptional regulation; when bound to D3, VDR regulates its own expression via VDREs in the gene’s enhancer region [24]. In addition, the VDR protein is stabilized by its ligand, extending the receptor’s halflife, which may in turn contribute to increased nuclear localization [24,27]. Expression of the VDR was previously identified in keloid fibroblasts, but has not been investigated in keloid epidermis. Expression of VDR was analyzed in uterine leiomyomas [28], which are benign fibrotic lesions with numerous similarities to keloids, including similarities in gene expression and high prevalence among African American women [29]. Western blot analysis demonstrated reduced VDR expression in over 60% of uterine fibroid tumors compared with adjacent normal myometrium [28]. Further, altered expression of VDR was observed in systemic sclerosis, a systemic connective tissue disorder that involves widespread fibrosis, including overproduction of collagen [30]. To determine if differences in VDR expression may be involved in keloid pathology, the current study investigated expression and nuclear localization of VDR in keloid scar tissue compared with normal skin.
2.
Materials and methods
2.1.
Collection of tissue samples
De-identified normal skin samples (N=24) were collected with University of Cincinnati (UC) Institutional Review Board (IRB) approval from breast or abdominal tissue from healthy donors undergoing elective plastic surgery procedures at the Shriners Hospitals for Children—Cincinnati and the UC Medical Center. Full thickness skin biopsies were collected after surgical excision from tissue designated as discard tissue. Because no protected health information (PHI) was collected from these donors, and only de-identified discard tissue was used, this was designated as “not human subjects research” by the UC IRB; therefore, patient consent was not required. For these deidentified samples, only patient age (in years), gender, and race/ethnicity were recorded. All skin samples were assigned a sequential identification number upon collection.
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Keloid scar patients were enrolled with informed consent in a study approved by the UC IRB. Patients were enrolled based on previously scheduled procedures for elective scar excision at the Shriners Hospitals for Children—Cincinnati, or the UC Medical Center. Biopsies of keloid scars were collected after scar excision from tissue designated as “discard” tissue. When possible (scars larger than 1cm2 in area), biopsies for histological sections were collected from the central region of each scar. The scars were identified as keloids based on clinical diagnosis by each patient’s physician and/or surgeon, which involved consideration of scar progression over time and expansion beyond the original wound boundary. Following keloid excision, diagnosis was confirmed by analysis of histological sections, including identification of keloidal collagen. Scars were excluded from the analysis if there was ambiguity in diagnosis. A total of 21 biopsies were collected from 19 distinct, unrelated donors. Two patients had multiple scars removed at different times after injury; these patients are indicated by asterisks in Supplemental Table 1. Patient demographic data collected for this study included date of birth, gender, and race/ethnicity for all patients. In addition, the date and mode of injury, the duration of the scar (e.g., time since the scar was first noticed or diagnosed as keloid), and any prior treatments, if known, were also recorded. In addition to keloid patients enrolled via informed consent, three additional keloid scars were collected as de-identified samples. For these scar samples, only the age, gender, and race/ethnicity of donors were recorded. Race/ethnicity classifications were based on patient selfidentification or, for younger patients, the guardian’s designation. Patients classified as “White” included non-Hispanic Whites/Caucasians. Patients classified as “Black” included those who self-identified as Black or African American, and included one patient who self-identified as “Black/mixed race.” Hispanic, Asian, and Native American patients were not included in the current study.
with an Eclipse 90i microscope and photographed with a DSRi1 Digital Microscope Camera (Nikon Instruments Inc., Melville, NY). Colorimetric immunostaining was used for quantitation of nuclei staining positive for VDR. Immunohistochemistry was performed using a different rabbit polyclonal anti-VDR antibody (Abcam; catalog #ab134826) that recognizes an internal sequence of the human vitamin D receptor. Detection utilized the Vectastain Elite ABC HRP Kit (Peroxidase, Universal; Vector Laboratories) followed by incubation (45s for all sections) with ImmPact NovaRed Peroxidase (HRP) Substrate (Vector Laboratories). Counterstaining of nuclei was performed by a brief (10s) incubation with Vector Hematoxylin QS (Vector Laboratories). Sections were all processed identically to minimize variability due to technical factors. For quantification of the percent of nuclei staining positive for VDR, three non-overlapping images per sample were photographed at 40X magnification (equivalent to 300 mm of epidermis). All nuclei were counted in each microscopic field, and all nuclei showing any level of positive staining for VDR were counted, enabling calculation of the percent of nuclei staining positive for VDR. Cells displaying any level of nuclear VDR-specific staining were counted as positive, even if there was little or no distinction between nuclear and cytoplasmic staining levels. For each tissue sample, two negative controls were performed on serial sections, omitting either the primary antibody or the biotinylated secondary antibody but including the hematoxylin nuclear stain. VDR-stained sections were carefully compared to negative control sections to distinguish between staining due to VDR immunolocalization and pigment due to epidermal melanin in some samples. For each field, the percent of nuclei staining positive for VDR (% VDR+) was determined, and a mean for each tissue sample was calculated.
2.2. Immunohistochemistry and quantification of nuclear localization
Statistical analyses were performed using SigmaPlot for Windows Version 11.0 (SyStat Software, Inc., San Jose, CA). Pairwise comparisons of numeric values were analyzed by ttest. Pairwise comparisons of categorical data were analyzed using the Chi-square test. Quantitative data are presented as meansstandard deviations. Differences were considered statistically significant at p<0.05.
Tissue biopsies were fixed in 10% buffered neutral formalin. Tissues were processed, including embedding in paraffin and sectioning, by the Histology Core Facility at Shriners Hospitals for Children—Cincinnati. Slides containing 4-mm thick paraffin sections were incubated for 2h at 55 C and deparaffinized by incubation in xylene followed by a graded alcohol series [31]. Antigen retrieval was performed using Citric Acid Based Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) and tissue was permeabilized in 0.05% Triton X-100 (Sigma–Aldrich, St. Louis, MO). For fluorescent labeling of VDR in tissue sections, a rabbit polyclonal anti-VDR antibody was used (Abcam, Cambridge, MA; catalog #ab3508). This antibody recognizes an epitope near the C-terminus of the human vitamin D receptor (amino acids 395-413). For fluorescent detection, Alexa Fluor 488conjugated Chicken Anti-rabbit IgG (H+L) was used as a secondary antibody (ThermoFisher Scientific, Waltham, MA; catalog #A-21441). Coverslips were mounted using 1 Vectashield Mounting Medium with DAPI (40 ,6-diamidino2-phenylindole; Vector Laboratories). Sections were viewed
2.3.
Statistical analyses
3.
Results
3.1.
Study population
Localization of VDR protein was analyzed in 24 normal skin samples and 24 keloid scar samples (Table 1). Normal skin donors ranged in age from 14 to 65 years (median age, 30.0 years); keloid scar patients ranged in age from 4 to 51 years (median 15.5 years). The mean age for normal skin donors was significantly older than the mean for keloid scar donors (31.5 13.3 years vs. 17.512.1 years, respectively; p<0.001). There was also a statistically significant difference in donor gender between the two groups, with one male and 23 females in the normal skin group, and 15 males and 9 females in the keloid
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Table 1 – Scar and skin samples used in study, and percent VDR-positive nuclei. Keloid scars: ID#
Ethnicity
Gender
Age (years)
Body site
%VDR+nuclei (meanstd. dev.)
753K 794K 795K 797K 798K 810K 817K 818K 829K 830K 835K 837K 850K 860K 868K 874K 878K 881K 884K 885K 897K 919K 934K 951K
W W B W W B W B B B B B W B B B B B B B Ba B B B
M M F M M M M M F M M F M M M M M M F F F F F F
11 17 20 10 17 13 8 16 12 17 17 4 16 9 13 22 17 37 51 10 8 10 15 51
Chin Chin Neck Ear Ear Arm Ear Shoulder Ear Neck Face Ear Face Face Chin Back Scapula Ear Neck Back Ear Ear Ear Breast
40.7 18.9 8.4 12.7 4.8 2.6 30.0 4.0 0.0 0.0 4.4 2.7 58.4 13.7 1.3 1.1 0.5 0.9 23.1 5.6 9.1 5.0 0.9 0.8 36.2 7.6 44.6 12.0 35.4 6.2 15.2 5.4 5.9 1.0 34.7 0.4 1.5 2.6 12.7 13.6 61.2 12.2 2.8 2.6 0.0 0.0 11.6 3.2
%VDR+nuclei (meanstd. dev.)
Normal skin ID#
Ethnicity
Gender
Age (years)
Body Site
822 824 831 844 858 859 861 863 865 866 869 870 876 880 882 886 894 896 898 916 920 925 927 929
W B W B W W W B B W B W B W W B W W B B B B B W
F F F F F F F F F F F F F F F M F F F F F F F F
42 24 38 38 42 38 40 55 52 30 24 30 18 15 65 17 14 26 25 25 20 30 31 17
Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Breast Abdomen Breast Abdomen Breast Breast Breast Breast Breast Breast
a
51.7 41.7 75.5 42.9 67.5 74.2 74.0 47.7 74.6 61.4 76.5 47.3 47.3 67.8 50.7 53.4 65.2 85.1 39.8 21.6 29.5 63.6 31.0 64.9
4.2 2.0 9.8 9.8 8.2 12.8 6.8 10.0 13.0 10.5 5.2 16.1 2.0 2.1 13.5 6.8 5.5 1.9 5.9 7.6 8.8 20.2 7.0 7.3
Abbreviations: W, white; B, Black or African American; F, female; M, male. Patient (897K) self-identified as African American and mixed race, but had light skin.
group (p<0.001). The normal skin group contained an equal number of black and white donors, whereas the keloid group had more black than white donors; however, the difference in racial makeup between the two groups was not statistically significant (p=0.136).
Normal skin samples were obtained primarily from breast tissue (22/24), with two samples obtained from abdominal tissue. In contrast, keloid scars were from a variety of body sites, including ear, face, chin, neck, back, scapula/shoulder, breast, and arm (Table 1). The mode of initial injury was known
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Fig. 1 – VDR localization in keloid scar and normal skin epidermis. VDR localization is shown in normal skin (A–I) and in keloid scars (J–R). Representative sections are shown. Fluorescent detection of VDR (green) is shown in the left column (A, D, G, J, M, P), nuclear staining with DAPI (blue) is shown in the center column (B, E, H, K, N, Q), and merged images are shown in the right
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Fig. 2 – Variable VDR staining and nuclear localization in normal skin and keloid scar epidermis from black and white donors. Shown are sections of normal skin (A–H) and keloid scar (I–P) from black (A–D, I–L) and white (E–H, M–P) donors. Donor identification numbers are indicated in each panel. For each pair of images, the first (A, C, E, G, I, K, M, O) shows colorimetric localization of VDR, and the second (B, D, F, H, J, K, N, P) shows a negative control section from the same donor processed without addition of primary anti-VDR antibody. Insets (G, O) show higher magnification images of boxed areas in these sections; examples of VDR-positive nuclei are indicated by arrowheads, and VDR-negative nuclei are indicated by arrows. The scale bar in A is the same for all panels: 50 mm.
for 19 of the 24 keloids (Supplemental Table 1). The majority (14/19) resulted from burns, and one keloid formed in an autograft donor site in a burn patient. Of the eight ear keloids with known etiologies, four occurred following ear piercing, and four were the result of burn.
3.2.
Expression and nuclear localization of VDR
Immunohistochemistry was initially performed using a fluorescent detection method. Comparisons among different samples from both keloid scar tissue and normal skin showed
variable levels of expression, both in dermal and epidermal VDR staining levels. Variability in overall expression levels was observed between groups, and among different donors within the same group. However, a striking difference was observed between normal skin and keloid scar epidermis: reduced nuclear localization of VDR protein was observed in keloid scar keratinocytes compared with normal skin (Fig. 1). In normal skin, bright staining of nuclei was clearly evident, with much lower levels of cytoplasmic staining observed (Fig. 1A–I). In contrast, staining for VDR in keloid scar epidermis was more diffuse, and although some nuclear localization was observed,
column (C, F, I, L, O, R). Insets (G–I and P–R) show higher magnification images of boxed areas in these sections; examples of VDRpositive nuclei are indicated by arrowheads, and VDR-negative nuclei are indicated by arrows. The scale bar in A is the same for all panels: 50 mm.
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this was patchy and levels were greatly reduced comparedwith normal skin (Fig. 1J–R). Expression levels and localization patterns varied among different keloid scar samples. Some had diffuse cytoplasmic staining with little distinction of staining levels between cytoplasm and nucleus, or with occasional nuclei showing increased VDR staining (Fig. 1J–O). In other keloids, epidermal VDR staining was mainly observed in the cytoplasm, with few nuclei positive for VDR signal (Fig. 1P–R). Positive VDR staining was observed in the dermal regions of both normal skin and keloid scar; expression levels were variable and no consistent differences between groups were observed. Collagen autofluorescence was observed in some samples, particularly in normal skin, complicating comparisons between groups using this staining technique. To confirm the specificity of the observed epidermal staining patterns, immunohistochemistry was performed using a second anti-VDR antibody directed at a different region of the VDR protein. Colorimetric immunostaining was performed using identical staining parameters for all samples, to enable comparisons of both localization and staining levels. Although variable staining levels were observed between samples from different individuals within each group, overall VDR expression levels were higher in normal skin compared with keloid scar epidermis (Fig. 2). Further, differences in nuclear localization of VDR were observed between normal skin and keloid scar epidermis. Nuclear localization was observed in all 24 normal skin
Fig. 3 – Quantitative analysis of VDR nuclear localization. The percent of epidermal nuclei staining positive for VDR (mean +standard deviation) is shown for normal skin and keloid scar samples (N=24 per group). The difference between groups was statistically significant (p<0.001).
samples, whereas some keloid scars showed little to no nuclear localization (Fig. 2). To quantify differences in VDR nuclear localization, the percent of epidermal nuclei showing positive localization of VDR was calculated for each sample (Table 1). For this quantitative analysis, only nuclear localization was considered; any level of VDR-specific staining in the nucleus was considered a positive signal. The percent of nuclei staining positive for VDR was variable in keloid scars, and no correlation with age, gender, body site, or area of keloid scar was found (Table 1, Supplemental Table 1 and data not shown). A comparison of all normal and keloid scar samples (N=24 each) showed that the percent of epidermal nuclei staining positive for VDR was significantly higher in normal skin compared with keloid scar (Fig. 3). To determine if skin color is associated with nuclear localization, analysis was performed on sub-groups based on the self-identified racial/ ethnic groups of donors. This analysis showed that normal epidermis of white donors displayed significantly higher levels of VDR nuclear localization compared with keloid scars from white donors; a significant difference was also observed between normal skin and keloid scar from black donors (Fig. 4). Interestingly, comparison of normal epidermis from black and white donors (N=12 each) revealed a significant difference in VDR nuclear localization, with higher levels in skin of white donors (Fig. 4). In keloid scars, no significant difference in nuclear localization was found between black (N=18) and white (N=8) donors; however, this analysis was not sufficiently powered to detect a possible significant difference due to the relatively low number of keloid scars from white donors.
Fig. 4 – Quantitative analysis of VDR nuclear localization in normal skin and keloid scar according to skin color. The percent of epidermal nuclei staining positive for VDR (mean +standard deviation) is shown for normal skin of white (N–W) and black (N–B) donors, and for keloid scars of white (K–W) and black (K–B) donors. Statistically significant differences are indicated (*p<0.001; # p<0.01).
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4.
Discussion
The results demonstrate significantly lower nuclear localization of VDR in keloid scar epidermis compared with normal skin. As a member of the nuclear hormone receptor superfamily, VDR activity involves transcriptional regulation of target genes. Therefore, reduced levels of nuclear localization may result in changes in the expression of downstream target genes. In systemic sclerosis, which is characterized by excessive deposition of ECM, VDR expression was reduced, and VDR knockdown was shown to enhance the fibrotic response of fibroblasts to TGF-b1 [30]. VDR expression was also reported to be reduced in many uterine fibroids, and treatment of uterine fibroid cells in vitro with D3 induced VDR expression and reduced expression of ECM genes [28]. Antifibrotic activity of D3 has been demonstrated in other organ systems and cell types, including lung, kidney, and liver [32–34]. D3 has been implicated in regulation of genes involved in EMT [35], a process implicated in fibrosis and cancer metastasis, as well as keloid scarring [11]. Knockdown or overexpression of VDR in a breast cancer cell line resulted in either increased or decreased expression of EMT markers, respectively [36]. Further, D3 acting via the VDR has been shown to exhibit immunomodulatory and anti-inflammatory properties in numerous cell types and disease models [37,38]. These previous studies suggest potential mechanisms whereby reduced VDR expression and nuclear localization may contribute to keloid pathology, by enhancing the responsiveness of cells to TGFb1 signaling and favoring EMT-like cellular changes and/or by modulating inflammation. However, increased TGFb1 expression in keloids may contribute to abnormal expression of VDR. Previous studies showed that persistent exposure of normal human dermal fibroblasts to TGF-b1 reduced VDR expression levels in vitro, suggesting complex crosstalk between these signaling pathways [30]. Further studies will be needed to determine the mechanism(s) whereby reduced VDR expression contributes to keloid pathology. In addition to differences in nuclear localization between keloid scars and normal skin, a significant difference was observed between normal skin samples from white and black donors. To our knowledge, this has not been previously reported. Although this result should be confirmed in a larger population, it suggests a possible mechanism for increased risk of keloid formation in skin of color. The basis for differential VDR localization in light vs. dark skin is unclear, but may be related to differences in vitamin D levels. African Americans have a greater incidence of vitamin D insufficiency compared to non-Hispanic whites [14,16,39,40]. Insufficiency results primarily from reduced vitamin D production in pigmented skin due to high levels of melanin, which shields keratinocytes from UVB [17]. Any alteration in the level of UVB reaching the skin impacts vitamin D production. In addition, diet plays an important role in vitamin D levels, and factors such as inadequate nutrition, cultural preferences, and lack of supplementation can influence vitamin D deficiency [16,41,42]. Vitamin D is known to regulate expression of VDR via VRDEs in the enhancer region of the VDR gene [24]. Hypothetically, vitamin D deficiency may contribute to reduced VDR expression or nuclear localization. Increased
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nuclear localization of VDR has been observed in response to D3 binding in vitro [43,44]. Previous studies demonstrated a two-fold increase in VDR protein levels in nuclear extracts from human skin treated topically with vitamin D in vivo [27]. This was due to reduced degradation upon ligand binding, which allowed VDR levels to increase in the nucleus [27]. This indicates that cells displaying reduced nuclear localization of VDR are still capable of responding to vitamin D by increasing VDR nuclear translocation and, hypothetically, increasing transcription of VDR target genes. This is relevant for the potential use of vitamin D as a therapeutic agent for abnormal scarring because it suggests the feasibility of modulating vitamin D-regulated gene expression in individuals with reduced basal nuclear VDR levels. This study had several limitations that resulted from our study design, which involved analysis of normal skin and keloid scar samples obtained from different donors. Because of this, the normal skin and keloid scar groups differed in several features including gender, age, and genetic background. Collection of a non-lesional skin biopsy from an individual predisposed to keloid scarring is likely to result in new keloid scar formation. This was of particular concern for the current study because the majority of keloid scar samples were collected from pediatric patients. Therefore, due to ethical considerations, we did not collect non-lesional skin biopsies from keloid patients for the purpose of this study. Whether samples were collected from de-identified tissue or from patients enrolled by informed consent, tissue was only used if it would otherwise be discarded. There were significant differences in age and gender between normal skin and keloid scar donors. The difference in age is due in part to the patient population, as samples were collected from patients at a pediatric burn specialty hospital as well as a medical center that treats predominantly adult patients. Normal skin samples were collected from elective breast reductions and abdominoplasties; the majority of these patients (19/24) were adults (>18 years of age). Most of the keloid scars were obtained from patients initially treated at a pediatric burn hospital, therefore the majority of these patients (19/24) were age 18 years or younger. However, the younger age range for keloid scars is also related to the prevalence of keloids in younger patients; keloid scars are most commonly observed in patients between the ages of 15 and 24 years [45]. Because the majority of plastic surgery procedures resulting in normal discard skin from healthy donors are performed in women, the majority of normal skin samples were from breast tissue collected from females. Although there were more male than female keloid patients, the distribution was not significantly different than a 50:50 distribution of males and females (data not shown). In contrast to the normal skin samples, keloid scars were from a variety of body sites. Although ears are among the most common sites for keloid formation [45], only nine of the keloids in the current study were ear keloids. Differences in body location may be relevant to VDR expression because VDR expression in skin is induced by UVB [46]. In the current study, the normal skin samples were from breast and abdominal skin, which are areas likely to be protected from UVB exposure. Thus, if body site influenced VDR expression due to sun exposure, the normal skin samples might be
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expected to show reduced expression compared with keloid samples from areas with greater potential for sun exposure. Vitamin D deficiency is common following major burn, and has been attributed to the inability to efficiently synthesize vitamin D in burned skin, metabolic alterations, lack of sun exposure during long hospitalizations, and inadequate supplementation [21,22,47]. A recent study found a correlation between vitamin D levels a year or more after extensive burn and scar formation [48]. Vitamin D levels were lower in patients with higher scores on the Modified Vancouver Scar Scale (MVSS), which indicates more severe scarring [48]. The authors speculated that patients with burn scars may have avoided sun exposure, leading to vitamin D deficiency [48]. This led them to recommend screening of patients with high MVSS scores for vitamin D deficiency. It would be interesting in future studies to determine whether vitamin D deficiency is predictive of abnormal scar formation.
Conflicts of interest The authors report no conflicts of interest.
Acknowledgments This work was supported by the Shriners Hospitals for Children (Medical Research Grant #85300). The authors thank the patients of the Shriners Hospital for Children—Cincinnati and the University of Cincinnati Medical Center who generously donated tissue samples for this study, and the surgeons and research nurses who assisted in collection of these samples. The authors also thank Kevin McFarland and Kelly Combs for critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. burns.2017.04.009. REFERENCES
[1] Stella M, Castagnoli C, Gangemi EN. Postburn scars: an update. Int J Low Extremity Wounds 2008;7:176–81. [2] Bock O, Schmid-Ott G, Malewski P, Mrowietz U. Quality of life of patients with keloid and hypertrophic scarring. Arch Dermatol Res 2006;297:433–8. [3] Van Loey NE, Van Son MJ. Psychopathology and psychological problems in patients with burn scars: epidemiology and management. Am J Clin Dermatol 2003;4:245–72. [4] Balci DD, Inandi T, Dogramaci CA, Celik E. DLQI scores in patients with keloids and hypertrophic scars: a prospective case control study. J Dtsch Dermatol Ges 2009;7:688–92. [5] Velez Edwards DR, Tsosie KS, Williams SM, Edwards TL, Russell SB. Admixture mapping identifies a locus at 15q21: 2– 22.3 associated with keloid formation in African Americans. Hum Genet 2014;133:1513–23. Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG. Hypertrophic scarring and keloids: pathomechanisms and
[6] current and emerging treatment strategies. Mol Med 2011;17:113–25. [7] Kiprono SK, Chaula BM, Masenga JE, Muchunu JW, Mavura DR, Moehrle M. Epidemiology of keloids in normally pigmented Africans and African people with albinism: population-based cross-sectional survey. Br J Dermatol 2015;173:852–4. [8] Shih B, Bayat A. Genetics of keloid scarring. Arch Dermatol Res 2010;302:319–39. [9] Brown JJ, Bayat A. Genetic susceptibility to raised dermal scarring. Br J Dermatol 2009;161:8–18. [10] Andrews JP, Marttala J, Macarak E, Rosenbloom J, Uitto J. Keloids: the paradigm of skin fibrosis—pathomechanisms and treatment. Matrix Biol 2016;51:37–46. [11] Hahn JM, McFarland KL, Combs KA, Supp DM. Partial epithelial-mesenchymal transition in keloid scars: regulation of keloid keratinocyte gene expression by transforming growth factor-ß1. Burns Trauma 2016;4:30. [12] Zhang GY, Cheng T, Luan Q, Liao T, Ni CL, Zheng X, et al. Vitamin D: a novel therapeutic approach for keloid, an in vitro analysis. Br J Dermatol 2011;164:729–37. [13] Bikle DD. Vitamin D and the skin. J Bone Miner Metab 2010;28:117–30. [14] Ginde AA, Liu MC, Camargo Jr. CA. Demographic differences and trends of vitamin D insufficiency in the US population, 1988–2004. Arch Intern Med 2009;169:626–32. [15] Engelman CD, Fingerlin TE, Langefeld CD, Hicks PJ, Rich SS, Wagenknecht LE, et al. Genetic and environmental determinants of 25-hydroxyvitamin D and 1,25dihydroxyvitamin D levels in Hispanic and African Americans. J Clin Endocrinol Metab 2008;93:3381–8. [16] Harris SS. Vitamin D and African Americans. J Nutr 2006;136:1126–9. [17] Holick MF. Vitamin D: a millenium perspective. J Cell Biochem 2003;88:296–307. [18] Armas LA, Dowell S, Akhter M, Duthuluru S, Huerter C, Hollis BW, et al. Ultraviolet-B radiation increases serum 25hydroxyvitamin D levels: the effect of UVB dose and skin color. J Am Acad Dermatol 2007;57:588–93. [19] Libon F, Cavalier E, Nikkels AF. Skin color is relevant to vitamin D synthesis. Dermatology 2013;227:250–4. [20] Wray CJ, Mayes T, Khoury J, Warden GD, Gottschlich M. The 2002 Moyer Award. Metabolic effects of vitamin D on serum calcium, magnesium, and phosphorus in pediatric burn patients. J Burn Care Rehabil 2002;23:416–23. [21] Gottschlich MM, Mayes T, Khoury J, Warden GD. Hypovitaminosis D in acutely injured pediatric burn patients. J Am Diet Assoc 2004;104:931–41 quiz. [22] Klein GL, Herndon DN, Chen TC, Kulp G, Holick MF. Standard multivitamin supplementation does not improve vitamin D insufficiency after burns. J Bone Miner Metab 2009;27:502–6. [23] Cooke GL, Chien A, Brodsky A, Lee RC. Incidence of hypertrophic scars among African Americans linked to vitamin D-3 metabolism? J Natl Med Assoc 2005;97:1004–9. [24] Saccone D, Asani F, Bornman L. Regulation of the vitamin D receptor gene by environment, genetics and epigenetics. Gene 2015;561:171–80. [25] Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ. The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer 2014;14:342–57. [26] Racz A, Barsony J. Hormone-dependent translocation of vitamin D receptors is linked to transactivation. J Biol Chem 1999;274:19352–60. [27] Li XY, Boudjelal M, Xiao JH, Peng ZH, Asuru A, Kang S, et al. 1,25-Dihydroxyvitamin D3 increases nuclear vitamin D3 receptors by blocking ubiquitin/proteasome-mediated degradation in human skin. Mol Endocrinol 1999;13:1686–94. [28] Halder SK, Osteen KG, Al-Hendy A. 1,25-Dihydroxyvitamin d3 reduces extracellular matrix-associated protein expression in human uterine fibroid cells. Biol Reprod 2013;89:150.
burns 43 (2017) 1506 –1515
[29] Luo X, Pan Q, Liu L, Chegini N. Genomic and proteomic profiling II: comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars. Reprod Biol Endocrinol 2007;5:35. [30] Zerr P, Vollath S, Palumbo-Zerr K, Tomcik M, Huang J, Distler A, et al. Vitamin D receptor regulates TGF-beta signalling in systemic sclerosis. Ann Rheum Dis 2014;74:e20. [31] Osborn M, Isenberg S. Immunocytochemistry of frozen and of paraffin tissue sections. In: Celis JE, editor. Cell biology: a laboratory handbook. San Diego, CA: Academic Press; 1998. p. 486–92. [32] Ramirez AM, Wongtrakool C, Welch T, Steinmeyer A, Zugel U, Roman J. Vitamin D inhibition of pro-fibrotic effects of transforming growth factor beta1 in lung fibroblasts and epithelial cells. J Steroid Biochem Mol Biol 2010;118:142–50. [33] Zhang Y, Kong J, Deb DK, Chang A, Li YC. Vitamin D receptor attenuates renal fibrosis by suppressing the renin-angiotensin system. J Am Soc Nephrol 2010;21:966–73. [34] Abramovitch S, Han-Bachar L, Sharvit E, Weisman Y, Ben TA, Brazowski E, et al. Vitamin D inhibits proliferation and profibrotic marker expression in hepatic stellate cells and decreases thioacetamide-induced liver fibrosis in rats. Gut 2011;60:1728–37. [35] Larriba MJ, Garcia de HA, Munoz A. Vitamin D and the epithelial to mesenchymal transition. Stem Cells Int 20162016: 6213872. [36] Pervin S, Hewison M, Braga M, Tran L, Chun R, Karam A, et al. Down-regulation of vitamin D receptor in mammospheres: implications for vitamin D resistance in breast cancer and potential for combination therapy. PLoS One 2013;8:e53287. [37] Lin Z, Li W. The roles of vitamin D and its analogs in inflammatory diseases. Curr Top Med Chem 2016;16:1242–61. [38] Vojinovic J. Vitamin D receptor agonists’ anti-inflammatory properties. Ann N Y Acad Sci 2014;1317:47–56.
1515
[39] Weishaar T, Vergili JM. Vitamin D status is a biological determinant of health disparities. J Acad Nutr Diet 2013;113:643–51. [40] Au LE, Harris SS, Dwyer JT, Jacques PF, Sacheck JM. Association of serum 25-hydroxyvitamin D with race/ethnicity and constitutive skin color in urban schoolchildren. J Pediatr Endocrinol Metab 2014;27:1095–100. [41] Macdonald HM. Contributions of sunlight and diet to vitamin D status. Calcif Tissue Int 2013;92:163–76. [42] Bailey RK, Fileti CP, Keith J, Tropez-Sims S, Price W, LisonOttey SD. Lactose intolerance and health disparities among African Americans and Hispanic Americans: an updated consensus statement. J Natl Med Assoc 2013;105:112–27. [43] Walters SN, Reinhardt TA, Dominick MA, Horst RL, Littledike ET. Intracellular location of unoccupied 1,25dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium. Arch Biochem Biophys 1986;246:366–73. [44] Yasmin R, Williams RM, Xu M, Noy N. Nuclear import of the retinoid X receptor, the vitamin D receptor, and their mutual heterodimer. J Biol Chem 2005;280:40152–60. [45] Trace AP, Enos CW, Mantel A, Harvey VM. Keloids and hypertrophic scars: a spectrum of clinical challenges. Am J Clin Dermatol 2016;17:201–23. [46] Demetriou SK, Ona-Vu K, Teichert AE, Cleaver JE, Bikle DD, Oh DH. Vitamin D receptor mediates DNA repair and is UV inducible in intact epidermis but not in cultured keratinocytes. J Invest Dermatol 2012;132:2097–100. [47] Klein GL, Chen TC, Holick MF, Langman CB, Price H, Celis MM, et al. Synthesis of vitamin D in skin after burns. Lancet 2004;363:291–2. [48] Terzi R, Guven M. Bone mineral density after burn injury and its relation to the characteristics of scar tissue. J Burn Care Res 2016;37:e263–7.