Vitamin D Receptor Expression in Vitiligo.

BACKGROUND: Vitiligo is a progressive depigmenting disorder characterized by loss of functional melanocytes from the epidermis. The etiopathogenesis of vitiligo is still unclear. Vitamin D has stimulatory effects on melanocytes and acts through its nuclear Vitamin D receptor (VDR) on target cells. AIMS AND
OBJECTIVES: The purpose of this study was to declare the role of Vitamin D in the pathogenesis of vitiligo.
MATERIALS AND METHODS: This case-control study included 30 vitiligo patients and 30 age, gender-matched healthy controls. Blood samples were withdrawn from the study subjects, and the serum 25(OH) D level was determined by an enzyme-linked immunosorbent assay technique. Serum 25(OH) D levels were divided into: Normal or sufficient (≥30 ng/ml), insufficient (< 30-> 20ng/ml), and deficient (≤20 ng/ml) levels. Skin biopsies were obtained from the depigmented lesions and clinically normal skin of vitiligo patients and from the controls, and VDR gene expression was determined using real-time polymerase chain reaction.
RESULTS: Only 10 patients with vitiligo (33.3%) had sufficient serum 25(OH) D levels (≥30 ng/ml), 12 patients (40%) had insufficient levels, and 8 patients (26.7%) had deficient levels. On the other hand, most of the controls (96.7%) had sufficient levels. The mean serum 25(OH) D level in patients was significantly decreased compared to controls (P < 0.001). The VDR-mRNA expression was also significantly decreased in lesional and nonlesional skin of patients compared to controls (P < 0.001, P < 0.001, respectively).
CONCLUSION: Vitamin D deficiency influences the extent of vitiligo and could contribute to the pathogenesis of vitiligo through its immunomodulatory role and its role in melanogenesis.

What was known?

Vitiligo is characterized by loss of functioning melanocytes

Vitamin D has stimulatory effects on melanocytes.

Introduction

Vitiligo is a common pigmentary disorder characterized by well-demarcated depigmented patches or macules of different shapes and sizes and caused by the destruction of functional melanocytes in the involved epidermis.[1]

Vitamin D is a steroid hormone that plays an important role in calcium homeostasis. The main source is an endogenous synthesis in the skin, which is induced by ultraviolet B (UVB) radiation.[2]

Vitamin D exerts its effect through a nuclear hormone receptor called the Vitamin D receptor (VDR).[3] Melanocytes express the VDRs, which take part in the regulation of melanin synthesis.[4] Several researchers pointed at the role of Vitamin D in melanogenesis. For instance, Tomita et al.,[5] found that Vitamin D3 increased the tyrosinase content of cultured human melanocytes and Watabe et al.[6] observed that 1,25(OH)2 D3 might stimulate the differentiation of immature melanocyte precursors.

The aim of this study was to evaluate Vitamin D role in the pathogenesis of vitiligo through the evaluation of Vitamin D serum level and VDR expression in tissue biopsies from the normal and vitiliginous skin.

Materials and Methods

This case-control study included 30 patients with vitiligo and 30 age and sex matched healthy controls. The patients and controls were of Fitzpatrick skin type IV recruited from individuals attending the outpatient clinic of the Beni-Suef University Hospitals in the period (from June 1, 2013 to September 1, 2013).

Exclusion criteria included the use of any drugs that could alter the outcome of the study such as topical or systemic Vitamin D or calcium, systemic steroids, weight loss drugs, cholesterol lowering drugs, thiazide diuretics, and sunscreens in the last 6 months.

Pregnant, lactating females, obese, smokers, subjects with malabsorption disorders, other autoimmune diseases, kidney diseases, and subjects receiving phototherapy the last 6 months were also excluded from the study.

Patient information was collected by one dermatologist including age, sex, type of vitiligo (generalized, localized or universal),[7] affected body surface area (BSA) according to the rule of nines,[8] and disease activity determined by the vitiligo disease activity (VIDA) score.[9]

The purpose of the study was explained to each patient, and written consent was taken. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institution's Human Research Committee.

Estimation of Vitamin D in serum using enzyme-linked immunosorbent assay

Blood samples were withdrawn and left to clot for 20 min, then centrifuged at 12,000 rpm for 10 min, then the separated serum was kept frozen till analysis. Serum samples were examined for 25(OH) D levels by enzyme-linked immunosorbent assay (ELISA) using the kit supplied by Immunodiagnostic USA. The samples were incubated with the detection antibody after the extraction step. Then peroxidase-conjugated antibody was added into the microplate well forming a complex of 25(OH) D - detection antibody - peroxidase conjugate. Tetramethylbenzidine was used as a substrate. The color density developed is proportional to Vitamin D concentration. Finally, to terminate the reaction, stop solution was added, and the microplate was read by ELISA reader at 450 nm.[10]

Serum levels of 25(OH) D were divided into sufficient (≥30 ng/ml); insufficient (20–30 ng/ml) and deficient (≤20 ng/ml) levels.[11]

Detection of Vitamin D receptor expression in tissue

Total RNA was isolated from skin tissue homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. The purity (A260/A280 ratio) and the concentration of RNA were obtained using spectrophotometry (GeneQuant 1300, Uppsala, Sweden). RNA quality was confirmed by gel electrophoresis.

First-strand cDNA was synthesized from 4 μg of total RNA using an Oligo (dT) 12–18 primer and Superscript™ II RNase reverse transcriptase, this mixture was incubated at 42°C for 1 h, the kit was supplied by SuperScript choice system (Life Technologies, Breda, The Netherlands).

Real-time quantitative polymerase chain reaction

Real-time polymerase chain reaction (RT-PCR) amplification was carried out using 10 μL amplification mixtures containing Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA), equivalent to 8 ng of reverse-transcribed RNA and 300 nM primers; the sequences of PCR primer pairs used for each gene are shown in Table 1. Reactions were run on an ABI PRISM 7900 HT detection system (Applied Biosystems); PCR reactions consisting of 95°C for 10 min (1 cycle), 94°C for 15 s, and 60°C for 1 min (40 cycles). Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1·7 Sequence detection software from PE Biosystems (Foster City, CA, USA). Relative expression of studied genes was calculated using the comparative threshold cycle method. All values were normalized to the GAPDH genes.[12]

Table 1

Primer sequences used for quantitative RT-PCR

Statistical analysis

Data were analyzed using the software, Statistical Package for Social Science, version 18. A frequency distribution with its percentage and descriptive statistics with mean and standard deviation (SD) were calculated. Chi-square, t-test, correlations were carried out whenever needed. P < 0.05 was considered significant.

Results

The gender ratio, age were not substantially different for each variable among patients with vitiligo (10 women, 20 men; mean ± SD age 32.5 ± 14.6 years), and healthy controls (14 women, 16 men; mean ± SD age 28 ± 5.7) (P = 0.4, P = 0.2, respectively). Clinical data of participants are presented in Table 2.

Table 2

Demographic data, clinical characteristics of vitiligo patients, and controls

Serum 25(OH) D Levels

The mean 25-hydroxy Vitamin D (25(OH) D) level in controls was almost twice that of patients (P < 0.001) [Figure 1].

Figure 1

Reduced mean level of 25(OH)D in vitiligo patients when compared to control subjects (P < 0.001)

We found that 10 patients with vitiligo (33.3%) versus almost all the controls (96.7%) had sufficient serum 25(OH) D levels; twelve patients (40%) versus only one control subject (3.3%) had insufficient levels; while eight patients (26.7%) and none of the controls had deficient 25(OH) D levels, which means that 20 patients (66.7%) versus only one control subject (3.3%) had 25(OH) levels below 30 ng/ml [Table 3 and Figure 2].

Table 3

Serum 25(OH)D levels and VDR expression in vitiligo patients, and controls

Figure 2

Among 25(OH)D levels were divided into: normal or sufficient (≥30 ng/ml), insufficient (< 30->20 ng/ml), and deficient (≤20 ng/ml) levels. Only 10 patient with vitiligo (33.3%) had sufficient serum 25(OH)D levels (≥30 ng/ml) while 20 patients (66.7%) had levels below 30 ng/ml

Although the levels of 25(OH) were low among patients, we could find no relation with the age, sex, disease duration, family history of vitiligo, stress at onset, vitiligo type, or VIDA score with P > 0.05 in all.

When we made a cut-off at serum 25(OH) D level of 30 ng/ml; we found that patients with 25(OH) D levels ≥30 ng/ml had higher affected BSA compared with those with levels < 30 ng/ml (mean ± SD; 47.5 ± 33.7 versus 33.3 ± 31.5, respectively).

The tissue Vitamin D receptor expression

The mean VDR expression in the tissue biopsies of controls was more than 8 times higher than the lesional tissue biopsies of patients (P < 0.001) and 3 times higher than the nonlesional biopsies from patients (P < 0.001).

Surprisingly, the mean VDR expression in the nonlesional biopsies was also more than double the mean VDR expression in the lesional biopsies (P < 0.001) [Table 3].

Again, we found no relation between patient age, sex, disease duration, family history, stress at onset, vitiligo type, VIDA score or affected BSA with the VDR expression (both in lesional and nonlesional tissue biopsies) and P > 0.05 in all.

No significant difference existed in VDR expression (in lesional or nonlesional tissue biopsies) between patient group with 25(OH) D levels above 30 ng/ml and the patient group below 30 ng/ml and the P > 0.05.

Discussion

The exact pathogenesis of vitiligo is unknown, but researchers suggested that an interplay of multiple factors as genetic, neural, oxidant-antioxidant, biochemical, minerals, and autoimmune process might induce vitiligo.[13]

Vitamin D is synthesized in the epidermal keratinocytes under the influence of UVB light.[14] Vitamin D compounds are known to influence melanogenesis[456] Moreover, topical Vitamin D analogs were used as monotherapy or in combination with phototherapy for the treatment of vitiligo.[15]

All the above mentioned prompted us to investigate the Vitamin D status in Egyptian vitiligo patients, and in order to do so we estimated the 25(OH) D levels in serum, which is a widely accepted indicator of overall Vitamin D status.[16] Serum 25(OH) D levels were divided into: Normal or sufficient (≥30 ng/ml), insufficient (<30->20 ng/ml), and deficient (≤20 ng/ml) levels.

Our results showed that the mean serum levels of 25(OH) D were markedly lower among cases than controls. We found that 26.7% of our patients had deficient 25(OH) levels. Moreover, 40% of patients had insufficient levels, which means that 66.7% of patients had 25(OH) D levels below 30 ng/ml.

Inconsistent with our results, Silverberg et al.[17] found that more than 68.9% of patients had serum levels of 25(OH) D below 30 ng/ml and, Saleh et al.[18] found that 97.5% of patients had deficient and also recommended levels of 25(OH) D.

On the contrary, Xu et al.[19] conducted a study over 171 Chinese patients and could detect no significant difference in the 25(OH) D levels between patients and controls.

The affected BSA was higher in patients with 25(OH) D level above 30 ng/ml compared to those with levels below 30 ng/ml, which means that the level of Vitamin D could influence the extent of the disease.

We propose that the low serum levels of Vitamin D in vitiligo patients could be a consequence of the disease, as well as a contributing factor, to the development of the disease.

As a consequence of the disease, patients suffering from vitiligo might have more psychological stress and tend not to expose their skin to the public, which would influence the Vitamin D synthesis.

On the other hand, deficient Vitamin D levels could contribute to the development of vitiligo through its immunomodulatory role, as well as its impact on melanogenesis.

A connection between some autoimmune diseases including type I diabetes mellitus, systemic lupus, psoriasis, and Vitamin D deficiency had been reported.[20] Besides, low levels of Vitamin D were also found in pemphigus vulgaris and bullous pemphigoid.[21]

The mechanism by which Vitamin D affects autoimmunity is not yet clear. It was found that 1,25(OH)2D3 could inhibit the differentiation and maturation of dendritic cells; reduce the expression of major histocompatibility complex class II, and inhibit the secretion of proinflammatory cytokines including interleukin 1 (IL-1), IL-2, IL-6, interferon gamma, tumor necrosis factor alpha, IL-12, and also inhibit IL-6 and IL-17 secretion thus impeding Th17 function.[19] Some of these proinflammatory and proapoptotic cytokines were found to play a role in the pathogenesis of vitiligo.[2223]

As a consequence, we could suggest that low levels of Vitamin D might impair Th1 and Th17 pathways contributing to the development of vitiligo.

As mentioned before, Vitamin D compounds influence maturation, differentiation, proliferation, migration, and melanin production of melanocytes; the melanocyte response to melanogenic stimuli is modulated by calcium-dependent regulatory enzymes or transcription factors, which might indicate that Vitamin D is likely to have an additional role in melanogenesis through its calcemic effect.

The VDR gene polymorphism was suggested to influence the Vitamin D level and consequently play an important role in the genetic susceptibility to vitiligo.[24] This primed us to investigate the VDR expression in vitiligo patients.

To the best of our knowledge, this is the first study to estimate the expression of VDR in vitiligo patients. Our results showed that the expression of VDR in tissue biopsies from the controls was significantly higher than both the lesional and nonlesional tissue biopsies from patients.

The low expression of VDR in vitiligo patients compared to controls could be explained by the markedly low Vitamin D levels that could influence the VDR expression on keratinocytes. On the other hand, the absent melanocytes, which harbor the VDR from vitiligo lesions, could simply explain the markedly higher VDR expression in controls compared to lesional tissue biopsies.

Surprisingly, we found no difference in VDR expression in patients with 25(OH) D levels above 30 ng/ml and below 30 ng/ml, which might point at the role of other factors like genetic suitability in influencing the VDR expression in vitiligo. However, these results have to be confirmed on a larger study group.

Recommendations

The screening of Vitamin D level for possible Vitamin D supplementation in patients of vitiligo before the starting of therapy is recommended and further studies that access the response to phototherapy in patients receiving Vitamin D supplementation are also recommended.

What is new?

Vitamin D levels are reduced in vitiligo patients

Vitamin D level influences the extent of vitiligo

Vitamin D receptor expression is reduced in vitiligo lesions compared to normal skin.