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Gene expression analysis of melanocortin system in vitiligo
Journal of Dermatological Science (2007) 48, 113—122
www.intl.elsevierhealth.com/journals/jods
Gene expression analysis of melanocortin system in vitiligo Ku ¨lli Kingo a,c, Eerik Aunin b,c, Maire Karelson a, Mari-Anne Philips b,c, Ranno Ra ¨tsep b,c, Helgi Silm a, ˜ks b,c,e,* Eero Vasar b,c, Ursel Soomets c,d, Sulev Ko a
Department of Dermatology and Venerology, University of Tartu, Estonia Department of Physiology, University of Tartu, Estonia c Centre of Molecular and Clinical Medicine, University of Tartu, Estonia d Department of Biochemistry, University of Tartu, Estonia e Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Estonia b
Received 18 April 2007; received in revised form 15 June 2007; accepted 18 June 2007
KEYWORDS Vitiligo; Melanocortin system; TRP1; DCT; mRNA expression
Summary Background: The melanocortin system in the skin coordinates pigmentation and immune response and could be implicated in the pathogenesis of vitiligo. Objectives: We aimed to analyze changes in expression of genes involved in skin pigmentation (melanocortin system and enzymes involved in melanin synthesis). Methods: With quantitative RT-PCR we measured the mRNA expression levels of eight genes from the melanocortin system and two enzymes involved in melanogenesis. RNA was extracted from both lesional and non-lesional skin of vitiligo patients and in nonsun-exposed skin of healthy subjects. Results: POMC (proopiomelanocortin) expression was lower in lesional skin compared to non-lesional skin. Expression of melanocortin receptors was increased in unaffected skin of vitiligo patients compared to healthy subjects and decreased in lesional skin compared to uninvolved skin of vitiligo patients, the differences were statistically significant in the cases of MC1R (melanocortin receptor 1) and MC4R (melanocortin receptor 4). TRP1 and DCT genes were down-regulated in lesional skin compared to non-lesional vitiligo skin or skin of healthy controls and up-regulated in uninvolved vitiligo skin compared to healthy control samples. In non-lesional skin, POMC expression was not elevated, possibly indicating that systemic influences are involved in up-regulation of MC receptor genes. Decreased expression of the analyzed genes in the lesional skin is not surprising, but statistically significant increased
* Corresponding author at: Department of Physiology, University of Tartu, 19 Ravila Street, 50411 Tartu, Estonia. Tel.: +372 7 374 335; fax: +372 7 374 332. E-mail address: [email protected] (S. Ko ˜ks). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.06.004
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K. Kingo et al. expression of studied genes in non-lesional skin from vitiligo patients is not described previously. Conclusion: In our mind, up-regulation of melanocortin system in non-lesional skin could be systemic compensation to restore normal pigmentation in lesions. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vitiligo is an acquired cutaneous disorder that presents with gradual skin depigmentation produced by the deterioration of melanocyte functions. Several hypotheses have proposed to explain the dysfunction and/or loss of melanocytes in epidermis of vitiligo patients [1,2]. These include an autoimmune mechanism, an auto-cytotoxic mechanism and an abnormality in melanocytes or in surrounding keratinocytes-producing factors necessary for the survival and function of melanocytes [3—5]. Till now the pathogenesis of vitiligo remains partially understood and probably involves various combinations of diverse mechanisms. The melanocortin system is an evolutionarily conserved regulatory module, operating as a coordinator and executor of responses to stress. The melanocortin system consists of the melanocortin peptides a-, b- and g-melanocyte-stimulating hormone (a-MSH, b-MSH and g-MSH) and adrenocorticotropic hormone (ACTH), that are post-translational products of the proopiomelanocortin (POMC) prohormone gene; the endogenous melanocortin receptor antagonists: agouti (ASIP) and agouti-related protein (AGRP); the family of five melanocortin receptors (MCRs) that exert the biological effects of the POMC peptides [6]. The components of the melanocortin system and their relationships are shown in Fig. 1. Described system is mainly expressed in the brain but also in many peripheral tissues including the skin. In the skin, the components of the melanocortin system are expressed in different cells. POMC mRNA is detected in the keratinocytes and melanocytes of normal epidermis and in the pilosebaceous unit [7]. Among POMC-derived peptides a-MSH and ACTH are the most abundant in the skin [8,9]. MC1R, the most strongly expressed melanocortin receptor in the human skin, has its highest expression in melanocytes [10]. The most active peptide for MC1R is a-MSH followed by ACTH, while b-MSH and g-MSH cause weak activation of this receptor [11]. The expression of MC2R has been detected in the melanocytes and adipocytes and this receptor is activated by ACTH [11,12]. MC4R, which is activated both by a-MSH and ACTH, has been reported to be expressed in dermal papilla cells [11,13]. The expression of MC5R has been established in sebocytes, adipocytes and skin mast
cells [12—14]. The binding of the POMC peptides to this receptor is similar to that of MC1R. No manifestation of MC3R mRNA in human skin has been reported. Functional purpose of the melanocortin system in the skin is to respond to external and internal stresses through local pigmentation, immune, epidermal, adnexal and vascular structures to stabilize skin function and prevent disruption of internal homeostasis [15,16]. Components of the melanocortin system are involved in determining skin and hair phenotypes as well as in different skin inflammatory disorders and malignancies. An association between the variability of the POMC gene and red hair phenotype has been demonstrated [17]. The abnormal expression of POMC peptides has been established in severe atopic dermatitis, psoriasis, scarring alopecia and inflammatory keloids as well as in the nodular type and metastatic melanomas and basal cell carcinoma [16]. The MC1R gene variants are linked with red hair and poor tanning ability [18—21]. Additionally, MC1R has been reported as a genetic
Fig. 1 The components of the melanocortin system: POMC, proopiomelanocortin; ASIP, agouti signalling protein; a-MSH, alpha-melanocyte-stimulating hormone; bMSH, beta-melanocyte-stimulating hormone; g-MSH, gamma-melanocyte-stimulating hormone; ACTH, adrenocorticotropic hormone; AGRP, agouti-related protein; MC1R, melanocortin receptor 1; MC2R, melanocortin receptor 2; MC3R, melanocortin receptor 3; MC4R, melanocortin receptor 4; MC5R, melanocortin receptor 5.
Melanocortin system expression in vitiligo
115 been studied, but association between variations of these genes and susceptibility to vitiligo has not been proved [30]. No studies have analyzed gene expression levels of the melanocortin system in vitiligo samples up to the present time.
2. Results Fig. 2 MC1R, MC2R, MC3R, MC4R and MC5R mRNA levels (relative to housekeeping gene HPRT mRNA level) in the skin from healthy controls (HCS).
risk factor for melanoma, basal cell carcinoma and squamous cell carcinoma [22,17]. ASIP inhibits the binding of a-MSH to MC1R, with resulting inhibition of melanogenesis [23,24]. Polymorphism in the ASIP gene at position 8818 was found to be associated with dark hair and brown eyes [25]. The role of AGRP in pigmentation has not been verified. As AGRP blocks a-MSH binding to melanocortin receptors, it is possibly implicated in human pigmentation [26]. The purpose of present study was to examine the expression variations of genes encoding mediators of the melanocortin system in non-lesional and lesional skin of vitiligo patients and in skin of healthy controls with the aim to explore the regulation of the cutaneous stress response system in vitiligo. In previous studies, a reduction in the level of the POMC peptide a-MSH has been demonstrated both in lesional skin and serum of vitiligo patients [27,28]. Moreover, Graham et al. demonstrated using a-MSH immunoreactive positive melanocytes that low expression of a-MSH in the lesional skin of vitiligo patients resulted from decreased expression of the peptide rather than a reduction in melanocyte numbers [29]. The relationship between vitiligo and MC1R and ASIP gene polymorphisms has also
Fig. 3 POMC mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. *p < 0.05.
Gene expression levels of POMC, the five melanocortin receptors (MC1R—MC5R) and endogenous melanocortin receptor antagonists (ASIP and AGRP) were measured by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) in punch biopsies from lesional and non-lesional skin of vitiligo patients (n = 31) and from non-sun-exposed skin of healthy subjects (n = 24). In addition, levels of two genes encoding enzymes concerned with melanogenesis — tyrosinase-related protein-1 (TRP1) and dopachrome tautomerase (DCT) — were measured. The presence of mRNA expression of examined genes in skin of healthy controls and vitiligo patients became evident in QRT-PCR with a relatively large number (27—36) of amplification cycles. In the samples, MC1R demonstrated the highest expression (amplification after 27 cycles), whereas the levels of MC2R, MC3R, MC4R and MC5R mRNAs were low (amplification after 32—36 cycles) (Fig. 2). POMC mRNA was detected at 28—32 cycles, ASIP and AGRP at 34—36 cycles. TRP1 and DCT mRNAs were detected at 28—30 cycles. None of the studied genes had statistically significant gender-related differences in mRNA expression levels. A difference in POMC mRNA expression level, although marginally satisfying the standard criteria of statistical significance ( p < 0.05), was evident between lesional and non-lesional skin of vitiligo subjects (Fig. 3). The POMC mRNA expression was 1.9 fold lower in involved skin compared with uninvolved skin in the vitiligo group. POMC expression was similar in uninvolved vitiligo skin and in skin biopsies of healthy controls. No significant differences in ASIP expression as well as AGRP expression were established when comparing the study groups (data not shown). Statistically significant differences in MC1R expression and MC4R expression between healthy controls and vitiligo patients were established. Specifically, MC1R mRNA expression was 1.6 fold higher in non-lesional skin of vitiligo patients when compared to healthy subjects ( p < 0.01; Fig. 4(a)). Likewise, the MC4R expression level in vitiligo non-lesional skin was 1.9 fold higher than in healthy skin ( p < 0.01; Fig. 4(b)). In lesional skin, 2.1 fold decrease of MC1R ( p < 0.0001; Fig. 4(a)) and 2.5 fold decrease of MC4R ( p < 0.01; Fig. 4(b)) expressions were detected
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Fig. 4 MC1R (a) and MC4R (b) mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. **p < 0.01 compared to healthy control; ++p < 0.01, +++p < 0.0001 compared to non-lesional skin.
when compared to non-lesional skin samples from patients with vitiligo. The mRNA expression levels of the three other melanocortin receptors (MC2R, MC3R and MC5R) were also increased in unaffected skin and decreased in lesional skin of vitiligo patients; however, the differences were not statistically significant (data not shown). Suppression of TRP1 and DCT expressions in lesional skin compared to non-lesional vitiligo skin and healthy controls was established. Specifically, 6.8 fold decrease of TRP1 mRNA expression in involved skin compared with skin of healthy subjects ( p < 0.0001; Fig. 5(a)) and 19.7 fold decrease of TRP1 in lesional skin compared to uninvolved skin in the vitiligo group ( p < 0.0001; Fig. 5(a)) was detected. The DCT mRNA expression was 7.6 fold lower in involved skin compared with skin of healthy controls ( p < 0.0001; Fig. 5(b)) and 12.9 fold lower in lesional skin compared to uninvolved skin in the vitiligo group ( p < 0.0001; Fig. 5(b)). Contrarily, the TRP1 mRNA expression was 2.9 fold higher in nonlesional skin of vitiligo patients compared to healthy controls ( p < 0.05; Fig. 5(a)). The DCT expression level in non-lesional skin of vitiligo patients showed
K. Kingo et al.
Fig. 5 TRP1 (a) and DCT (b) mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. *p < 0.05, *** p < 0.0001 compared to healthy control; +++p < 0.0001 compared to non-lesional skin.
also a clear tendency of increase when compared to healthy subjects while not reaching the level of statistical significance ( p = 0.14). No differences in the expressions of genes of the melanocortin system and genes involved in melanin synthesis were detected between subgroups of vitiligo based on the extent of involvement and progression of the disease. Possible interactions between the expressions of the studied genes were examined by Spearman rank correlation. In addition, the correlations between the expressions of genes of the melanocortin system and tyrosinase (TYR), essential enzyme in melanin synthesis, were examined. In our previous study, a statistically significant decrease in TYR mRNA expression was observed in lesional skin compared to non-lesional skin of vitiligo patients and in skin of healthy subjects (both p < 0.0001) [31]. TYR expression was elevated in uninvolved vitiligo skin compared to skin samples from controls, but this difference was not statistically significant. The data of the correlations between the expressions of the genes of the melanocortin system in the control group and patient group are presented in Table 1.
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0.57 /0.22/0.27 0.29/0.05/0.17 0.21/0.11/0.29 0.01/0.15/0.45 * 0.60*/0.54**/0.60 ** 0.79***/0.79***/0.83 *** 1/1/1 0.36/0.51**/0.62 *** 0.54 /0.28/0.24 0.11/0.15/0.36 0.29/0.27/0.16 0.02/0.09/0.17 0.60*/0.63***/0.44 * 1/1/1 0.79***/0.79***/0.82 *** 0.37/0.26/0.76 *** 0.07/0.31/ 0.28/0.052/0.23 0.27/0.02/0.24 0.43/0.29/0.30 1/1/1 0.60*/0.63***/0.44 * 0.60*/0.54**/0.60 ** 0.41/0.20/0.28 0.1/0.07/0.15 0.73***/0.27/0.17 0.06/0.09/0.36 1/1/1 0.43/0.29/0.30 0.02/0.09/0.23 0.01/0.15/0.45 * 0.13/0.21/0.24 0.05/0.14/0.03 0.01/0.04/0.12 1/1/1 0.06/0.09/0.36 0.27/0.02/0.24 0.29/0.27/0.16 0.21/0.11/0.29 0.35/0.28/0.01
0.32/0.42 /0.16 1/1/1 0.01/0.04/0.12 0.73***/0.27/0.17 0.28/0.05/0.23 0.11/0.15/0.36 0.29/0.05/0.17 0.21/0.35/0.29 p < 0.05. p < 0.01. p < 0.001. ***
*
**
1/1/1 0.32/0.42*/0.16 0.05/0.14/0.02 0.11/0.07/0.15 0.07/0.31/0.29 0.54*/0.28/0.24 0.57**/0.22/0.27 0.41/0.12/0.26 POMC ASIP AGRP MC1R MC2R MC3R MC4R MC5R
MC5R (HCS/NLS/LS) MC4R (HCS/NLS/LS)
** *
MC3R (HCS/NLS/LS) MC2R (HCS/NLS/LS) MC1R (HCS/NLS/LS) AGRP (HCS/NLS/LS) ASIP (HCS/NLS/LS) POMC (HCS/NLS/LS)
Thehypothalamic—pituitary—adrenalaxis(HPA)inthe brain is the main mediator of the systemic response to stress.TheskinexpressesanequivalentoftheHPAaxis: a cutaneous defence system that operates as a coordinator and executor of local responses to stress. The melanocortin system in the skin is a part of the cutaneous HPA axis [12]. In the present study, we analyzed expression of melanocortin system genes in skin samples from patients with vitiligo. Our study sample contains 25 patients with generalized vitiligo, 1 with segmental vitiligo, 4 with local form and 1 with universal form. As segmental vitiligo and generalized vitiligomayhavedifferentpathogenesis,wecompared meanexpressionsofstudiedgenesindifferentformsof vitiligo. There was no significant difference in the expression pattern and we decided to merge the expression results of different forms (segmental, generalized, localized and universal) of vitiligo into one study group. Genes of the melanocortin system analyzed in the human skin in the present study were proopiomelanocortin (POMC), five melanocortin receptors (MC1R—MC5R), agouti signalling protein (ASIP) and agouti-related protein (AGRP). Our results reaffirmed the expression of cutaneous stress response system in human skin, whereas expression of MC3R mRNA in human skin has not previously been reported. Further studies using histochemical methods must be conducted to localize MC3R expression to distinct cell types in the skin. The expression profiles of all five melanocortin receptors were analogous between the study groups, a markedly significant difference between lesional and nonlesional vitiligo skin ( p < 0.0001) was established only in the case of MC1R. Probably, the expression levels of the genes indicate their functional relevance. In our samples, MC1R demonstrated the highest expression, while the rest of the melanocortin
Table 1 Spearman rank correlation coefficient (Spearman r) for expression values of melanocortin system
3. Discussion
*
Variables of the mRNA expression of different components of the melanocortin system were positively related. Among the expressions of TYR, TRP1 and DCT mRNAs, a strong positive correlation equally in skin of healthy controls and in skin of vitiligo patients was observed (r > 0.70; p < 0.0001; Fig. 6(a—c)). Further, positive correlation was noted between the levels of MC1R and TRP1 in skin of healthy controls (r = 0.47; 95% CI 0.001—0.77; p < 0.05; Fig. 7(a)). Contrarily, no statistically significant correlation between variables of MC1R mRNA and expressions of TYR, TRP1 and DCT mRNAs were observed in non-lesional and lesional vitiligo skin (Fig. 7(b and c)).
0.41/0.12/0.26 0.21/0.35/0.29 0.35/0.28/0.01 0.13/0.21/0.24 0.41/0.20/0.28 0.37/0.26/0.76 *** 0.36/0.51**/0.62 *** 1/1/1
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Fig. 6 (a) The relation of the levels of TYR (TYR_HCS), TRP1 (TRP1_HCS) and DCT (DCT_HCS) in skin of healthy subjects. (b) The relation of the levels of TYR (TYR_NLS), TRP1 (TRP1_NLS) and DCT (DCT_NLS) in non-lesional vitiligo skin. (c) The relation of the levels of TYR (TYR_LS), TRP1 (TRP1_LS) and DCT (DCT_LS) in lesional vitiligo skin.
receptors had relatively low expression levels. Moreover, the correlation analysis presented a moderate up to strong positive relation between the levels of mRNA of the different melanocortin receptors. High homology in sequences may be the cause of the observed similar expression profile of the melanocortin receptors [11]. Alternatively, this kind of expression profile of the melanocortin receptors could be explained by concomitant expression of these genes. Concomitant expression of MC1R and MC2R has been reported in human keratinocytes and epidermal melanocytes [32—34]. Concomitant expression of MC1R and MC4R has been observed in human dermal papilla cells [35]. In the skin, the potential role of the melanocortin system in the network of cutaneous inflammation and pigmentation has been verified. The possible role of markers of the melanocortin system in the pathogenesis of vitiligo remains unclear. In the present study, POMC mRNA expression was found to be lower in lesional skin compared with non-lesional
skin in the vitiligo group ( p < 0.05). At the same time, we did not detect difference in the expression of POMC between non-lesional skin from vitiligo patients and skin from healthy controls. The mRNA expression levels of the melanocortin receptors, mediating the effects of POMC peptides, were decreased in lesional skin compared to uninvolved skin and increased in unaffected skin of vitiligo patients compared to healthy subjects, the differences were statistically significant ( p < 0.01) in the cases of MC1R and MC4R. Decreased expression of MC1R in lesional skin is not surprising, because this fits the loss of functional melanocytes–—major cell type expressing MC1R. Contrarily, MC4R is expressed only in dermal papilla cells. Similar down-regulation of MC1R and MC4R in lesional skin may occur due to high homology in their sequences, which may provide similar regulatory regions employed in lesional skin. Increased expression of MC receptors in nonlesional skin of patients is not so well understood. This is not caused by natural UVR exposure, because
Melanocortin system expression in vitiligo
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Fig. 7 (a) The relation of the levels of MC1R (MC1R_HCS) and TYR (TYR_HCS), TRP1 (TRP1_HCS) and DCT (DCT_HCS) in skin of healthy controls. (b) The relation of the levels of MC1R (MC1R_NLS) and TYR (TYR_NLS), TRP1 (TRP1_NLS) and DCT (DCT_NLS) in non-lesional vitiligo skin. (c) The relation of the levels of MC1R (MC1R_LS) and TYR (TYR_LS), TRP1 (TRP1_LS) and DCT (DCT_LS) in lesional vitiligo skin.
skin biopsies were taken from truly non-exposed areas. Also, distribution of skin phototypes between study groups was balanced. Over-expression of MC receptors possibly indicates the existence of compensation system to restore normal pigmentation in lesions. Lack of difference in case of POMC in the non-lesional and control skin samples suggest that this over-expression is induced by systemic, circulating influences rather than local ones. Tyrosinase (TYR) and tyrosinase-related proteins (TRP1 and DCT) are closely related melanocytespecific gene products involved in melanogenesis and in the biology of melanocytes. Down-regulation of TYR mRNA expression has been documented in lesional skin of vitiligo patients [36]. Accordingly, in our previous study, a statistically significant decrease in TYR expression was observed in lesional skin compared to non-lesional skin of vitiligo patients and healthy control subjects (both p < 0.0001) [31]. In addition, significantly lower level of TRP1 mRNA expression by vitiligo melanocytes compared to control normal melanocytes is established [37]. No expression of DCT mRNA has
been investigated in vitiligo up to the present time. In the present study, suppression of expressions of TRP1 and DCT mRNAs, as verification of decreased melanin synthesis in vitiligo, was established in lesional skin of vitiligo patients. Current literature indeed suggests that the lesional area is characterized by the absence of melanocytes or by the presence of ineffective melanocytes. However, not all melanocytes are destroyed in lesions and this explains why these mRNAs are still in detectable level. We found expressional up-regulation of genes involved in melanin synthesis in uninvolved skin samples of vitiligo patients compared to healthy control samples. In case of TRP1, this difference was statistically significant. As we also found overexpression of melanocortin receptors in the same samples, activation of enzyme genes in non-lesional skin is a consequence of melanocortin stimulation. This indicates functional relevance of our finding. It is possible that the suppression of melanin production activates melanocortin receptors transcriptionally through negative feedback to restore normal
120 pigmentation. On the other hand, melanocortins have the widest spectrum of immunomodulatory/ anti-inflammatory capacities [38—41]. Increased expressions of pro-inflammatory cytokines (IL-2, TNF-a, IL-6 and INF-g) in lesional skin compared to uninvolved skin of vitiligo patients have been reported [42,43]. Up-regulation of the melanocortin receptors in non-lesional skin, established in the present study, may attempt to inhibit the production or action of proinflammatory factors and thereby make an effort to suppress inflammatory responses in lesional skin of vitiligo patients. Positive correlation between the levels of MC1R and TRP1 was found in the skin of healthy controls, this correlation did not exist in patients. Described finding indicates that significant changes in transcriptional regulation of the melanocortin and melanin synthesis system occur in skin during vitiligo. In conclusion, the expression of genes of the melanocortin system is altered in vitiligo. Decreased expression of the genes of the melanocortin system and enzymes of melanogenesis in the lesional skin demonstrated in the present study is not surprising and fits with existing data. On the other hand, statistically significant increased expression in non-lesional skin from vitiligo patients is not described previously. In our mind, this up-regulation could be systemic compensation to restore normal pigmentation in lesions.
4. Materials and methods The study protocols and informed consent forms were approved by the Ethical Review Committee on Human Research of the University of Tartu. All participants signed a written informed consent. The patients and control subjects in the study were Caucasians living in Estonia. Unrelated patients with vitiligo (n = 31; 22 females; 9 males; age range 22—75 years) from the Department of Dermatology, the University of Tartu, were included in the study. The mean age of vitiligo onset of the patients was 30.0 years and the mean duration of vitiligo was 19.2 years. Five patients had a family history of vitiligo. None of the patients included in the study had received specific therapy in the previous 6 months. The clinical signs on which the diagnosis of vitiligo was based on were characteristic loss of skin pigmentation with typical localization and white colour on the skin lesions under Woods lamp. The type of vitiligo was based on the extent of involvement and the distribution of pigmentation. Focal vitiligo involves depigmentation in a localized, non-dermatomal distribution (n = 4(F)). Segmental vitiligo encompasses depigmentation of the dermatomal,
K. Kingo et al. asymmetric distribution (n = 1(F)). Generalized vitiligo is characterized by bilateral, symmetric loss of pigmentation of the torso, face, neck, or extensor surfaces of the hands and legs (n = 25; 17(F); 8(M)). Universal vitiligo occurs as depigmentation of the entire body surface area (n = 1(M)). The stage of vitiligo was based on the interval of manifestation of new areas of depigmentation or enlargement of the area of depigmentation. The patients were divided into two subgroups based on the stage of progression of the disorder: patients with progressive vitiligo (active vitiligo, in which new areas of depigmentation or enlargement of depigmentation were observed during the previous 3 months; n = 22; 16(F); 6(M)) and patients with stable vitiligo (inactive vitiligo, in which no new depigmentation or enlargement of depigmentation had been observed for more than 3 months; n = 9; 6(F); 3(M)). The control group consisted of healthy volunteers (n = 24; 17 female; 7 male; age range 21—67 years) free from the positive family history of vitiligo and other chronic dermatoses. Control subjects were recruited from health care personnel, medical students and patients present at the dermatological outpatient clinic with either facial teleangiectasis or skin tags. Two skin biopsies (Ø 4 mm) were obtained from each patient with vitiligo: one from the central part of involved skin and another from non-sun-exposed uninvolved skin. One skin biopsy (Ø 4 mm) from nonsun-exposed skin was taken from healthy control subjects. The non-sun-exposed skin was defined as the skin never exposed to UVR previously and definitely not exposed to natural UVR in the last 12 months. Biopsies from uninvolved skin of vitiligo patients and healthy controls were taken from the lower abdomen. All probands had skin phototype II (9 controls, 13 patients) or III (15 controls, 18 patients), Fitzpatrick classification [44]. Biopsies were instantaneously snap-frozen in liquid nitrogen and stored at 80 8C until further use. Total RNA was isolated from tissues using RNeasy Fibrous Tissue Mini Kit (QIAGEN Sciences, MD, USA) following the protocol suggested by the manufacturer. For tissue homogenization, Ultra-Turrax T8 homogenizer (IKA Labortechnik, Germany) was used. RNA was dissolved in RNase free water and stored until further use at 80 8C. For each RT-PCR reaction, 500 ng of total RNA was converted into cDNA. The reverse transcription reactions were performed with a reverse transcriptase (SuperScript III; Invitrogen Corp., Carlsbad, CA, USA) and poly(T18) oligonucleotides in accordance with the manufacturer’s instruction. The reaction mixtures were incubated at 65 8C for 5 min, at 0 8C for 1 min, at 50 8C for 90 min, at 70 8C for 5 min and finally stored at 80 8C.
Melanocortin system expression in vitiligo Gene expression levels were detected in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Reactions were carried out in 10 ml reaction volumes in four replicates. The expression levels of AGRP, ASIP, the melanocortin receptor genes and TRP1 and DCT were detected applying TaqMan-QRT-PCR method using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). For the detection of the expression levels of AGRP, ASIP, MC3R, MC4R, MC5R, TRP1 and DCT, we used TaqMan Assay-OnDemand FAM-labelled MGB-probe gene expression assay mixes (20X, Applied Biosystems, Foster City, CA, USA). The assay mixes used were Hs00361403_g1 (AGRP), Hs00181770_m1 (ASIP), Hs00252036_s1 (MC3R), Hs00271877_s1 (MC4R), Hs00271882_s1 (MC5R), Hs00167051_m1 (TRP1) and Hs00157244_ m1 (DCT). For the detection of the expression levels of MC1R and MC2R, we used gene-specific primers (MC1R: forward 50 -TGCGGCTGCATCTTCAAG-30 , reverse 50 -TGATGGCATTGCAGATGATGA-30 ; MC2R: forward 50 -CTCGATCCCACACCAGGAA-30 , reverse 50 TGTGATGGCCCCTTTCATGT-30 ) and MGB-labelled probe (MC1R: FAM-TTCAACCTCTTTCTCGCC-NFQ; MC2R: FAM-TCTCCACCCTCCCCAGA-NFQ). For the detection of HPRT-1 (hypoxanthine phosphoribosyltransferase-1) expression level, gene-specific primers (HPRT-1 exon 6, 50 -GACTTTGCTTTCCTTGGTCAGG-30 ; HPRT-1 exon 7, 50 -AGTCTGGCTTATATCCAACACTTCG-30 ; final concentrations 300 nM) and VIC-TAMRA-labelled probe (VIC-50 -TTTCACCAGCAAGCTTGCGACCTTGA-30 -TAMRA; final concentration 200 nM) were used. The expression level of POMC was detected using qPCR Core Kit for SYBR Green I (Eurogentec, Seraing, Belgium) and gene-specific primers (forward 50 CTACGGCGGTTTCATGACCT-30 , reverse 50 -CCCTCACTCGCCCTTCTTG-30 , final concentrations 100 nM). For quantification of mRNA, we used comparative Ct method (DCt value), where the amount of target transcript was normalized according to the level of endogenous reference HPRT-1 (hypoxanthine phosphoribosyl-transferase-1). Adjustment to normal distribution was tested by the Kolmogorov—Smirnov test. The distribution of measurements of gene expressions by the applied method did not follow a Gaussian distribution. Mann—Whitney U-test and Kruskal—Wallis test were used to test for differences between the groups using GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA). Correlation analysis was used to investigate relations between two parameters of one group. For measure of correlation, the Spearman rank correlation was applied. For all tests, a p value < 0.05 was considered significant.
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Acknowledgements This study was financially supported by the targetbased funding from the Estonian Ministry of Education Grant No. SF0180043s07, by University of Tartu Research Grant PARFS 05901, by the Estonian Science Foundation Grants No. 6576, 5712 and 5688 and by the Centre of Molecular and Clinical Medicine Grant VARMC-TIPP.
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122 [16] Slominski A, Wortsman J, Mazurkiewicz JE, Matsuoka L, Dietrich J, Lawrence K, et al. Detection of proopiomelanocortin-derived antigens in normal and pathologic human skin. J Lab Clin Med 1993;122:658—66. [17] Bastiaens MT, ter Huurne JA, Kielich C, Gruis NA, Westendorp RG, Vermeer BJ, et al. Melanocortin-1 receptor gene variants determine the risk of nonmelanoma skin cancer independently of fair skin and red hair. Am J Hum Genet 2001;68:884—94. [18] Valverde P, Healy E, Jackson I, Rees JL, Thody AJ. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat Genet 1995;11:328—30. [19] Krude H, Biebermann H, Luck W, Horn R, Brabant G, Gruters A. Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat Genet 1998;19:155—7. [20] Healy E, Flannagan N, Ray A, Todd C, Jackson IJ, Matthews JN, et al. Melanocortin-1-receptor gene and sun sensitivity in individuals without red hair. Lancet 2000;355:1072—3. [21] Flanagan N, Healy E, Ray A, Philips S, Todd C, Jackson IJ, et al. Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation. Hum Mol Genet 2000;9:2531—7. [22] Kennedy C, ter Huurne J, Berkhout M, Gruis N, Bastiaens M, Bergman W, et al. Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color. J Invest Dermatol 2001;117:294—300. [23] Suzuki I, Tada A, Ollmann MM, Barsh GS, Im S, Lamoreux ML, et al. Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin. J Invest Dermatol 1997;108:838—42. [24] Voisey J, Kelly G, Van Daal A. Agouti signal protein regulation in human melanoma cells. Pigm Cell Res 2003;16:65—71. [25] Kanetsky PA, Swoyer J, Panossian S, Holmes R, Guerry D, Rebbeck TR. A polymorphism in the agouti signaling protein gene is associated with human pigmentation. Am J Hum Genet 2002;70:770—5. [26] Yang YK, Thompson DA, Dickinson CJ, Wilken J, Barsh GS, Kent SB, et al. Characterization of agouti-related protein binding to melanocortin receptors. Mol Endocrinol 1999;13: 148—55. [27] Thody AJ, Ridley K, Penny RJ, Chalmers R, Fisher C, Shuster S. MSH peptides are present in mammalian skin. Peptides 1983;4:813—6. [28] Pichler R, Sfetsos K, Badics B, Gutenbrunner S, Aubock J. Vitiligo patients present lower plasma levels of alpha-melanotropin immunoreactivities. Neuropeptides 2006;40: 177—83. [29] Graham A, Westerhof W, Thody AJ. The expression of alphaMSH by melanocytes is reduced in vitiligo. Ann NY Acad Sci 1999;885:470—3.
K. Kingo et al. [30] Na GY, Lee KH, Kim MK, Lee SJ, Kim DW, Kim JC. Polymorphisms in the melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) genes in Korean vitiligo patients. Pigm Cell Res 2003;16:383—7. [31] Kingo K, Philips MA, Aunin E, Luuk H, Karelson M, Ratsep R, et al. MYG1, novel melanocyte-related gene, has elevated expression in vitiligo. J Dermatol Sci 2006;44:119—22. [32] Curry JL, Pinto W, Nickoloff BJ, Slominski AT. Human keratinocytes express functional alpha-MSH (MC1-R) receptors. In Vitro Cell Dev Biol Anim 2001;37:234—6. [33] Moustafa M, Szabo M, Ghanem GE, Morandini R, Kemp EH, MacNeil S, et al. Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alphamelanocyte stimulating hormone and adrenocorticotropic hormone peptides. J Invest Dermatol 2002;119:1244—53. [34] Suzuki I, Cone RD, Im S, Nordlund J, Abdel-Malek ZA. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 1996;137:1627—33. [35] Bohm M, Luger TA, Tobin DJ, Garcia-Borron JC. Melanocortin receptor ligands: new horizons for skin biology and clinical dermatology. J Invest Dermatol 2006;126:1966—75. [36] Machado Filho CD, Almeida FA, Proto RS, Landman G. Vitiligo: analysis of grafting versus curettage alone, using melanocyte morphology and reverse transcriptase polymerase chain reaction for tyrosinase mRNA. Sao Paulo Med J 2005;123:187—91. [37] Jimbow K, Chen H, Park JS, Thomas PD. Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinase-related protein in vitiligo. Br J Dermatol 2001;144:55—65. [38] Luger TA, Scholzen TE, Brzoska T, Bohm M. New insights into the functions of alpha-MSH and related peptides in the immune system. Ann NY Acad Sci 2003;994:133—40. [39] Wintzen M, Gilchrest BA. Proopiomelanocortin, its derived peptides, and the skin. J Invest Dermatol 1996;106:3—10. [40] Teofoli P, Motoki K, Lotti TM, Uitto J, Mauviel A. Propiomelanocortin (POMC) gene expression by normal skin and keloid fibroblasts in culture: modulation by cytokines. Exp Dermatol 1997;6:111—5. [41] Wintzen M, Yaar M, Burbach JP, Gilchrest BA. Proopiomelanocortin gene product regulation in keratinocytes. J Invest Dermatol 1996;106:673—8. [42] Caixia T, Hongwen F, Xiran L. Levels of soluble interleukin-2 receptor in the sera and skin tissue fluids of patients with vitiligo. J Dermatol Sci 1999;21:59—62. [43] Grimes PE, Morris R, Avaniss-Aghajani E, Soriano T, Meraz M, Metzger A. Topical tacrolimus therapy for vitiligo: therapeutic responses and skin messenger RNA expression of proinflammatory cytokines. J Am Acad Dermatol 2004;51: 52—61. [44] Fitzpatrick TB, Miyamoto M, Ishikawa K. The evolution of concepts of melanin biology. Arch Dermatol 1967;96:305—23.
www.intl.elsevierhealth.com/journals/jods
Gene expression analysis of melanocortin system in vitiligo Ku ¨lli Kingo a,c, Eerik Aunin b,c, Maire Karelson a, Mari-Anne Philips b,c, Ranno Ra ¨tsep b,c, Helgi Silm a, ˜ks b,c,e,* Eero Vasar b,c, Ursel Soomets c,d, Sulev Ko a
Department of Dermatology and Venerology, University of Tartu, Estonia Department of Physiology, University of Tartu, Estonia c Centre of Molecular and Clinical Medicine, University of Tartu, Estonia d Department of Biochemistry, University of Tartu, Estonia e Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Estonia b
Received 18 April 2007; received in revised form 15 June 2007; accepted 18 June 2007
KEYWORDS Vitiligo; Melanocortin system; TRP1; DCT; mRNA expression
Summary Background: The melanocortin system in the skin coordinates pigmentation and immune response and could be implicated in the pathogenesis of vitiligo. Objectives: We aimed to analyze changes in expression of genes involved in skin pigmentation (melanocortin system and enzymes involved in melanin synthesis). Methods: With quantitative RT-PCR we measured the mRNA expression levels of eight genes from the melanocortin system and two enzymes involved in melanogenesis. RNA was extracted from both lesional and non-lesional skin of vitiligo patients and in nonsun-exposed skin of healthy subjects. Results: POMC (proopiomelanocortin) expression was lower in lesional skin compared to non-lesional skin. Expression of melanocortin receptors was increased in unaffected skin of vitiligo patients compared to healthy subjects and decreased in lesional skin compared to uninvolved skin of vitiligo patients, the differences were statistically significant in the cases of MC1R (melanocortin receptor 1) and MC4R (melanocortin receptor 4). TRP1 and DCT genes were down-regulated in lesional skin compared to non-lesional vitiligo skin or skin of healthy controls and up-regulated in uninvolved vitiligo skin compared to healthy control samples. In non-lesional skin, POMC expression was not elevated, possibly indicating that systemic influences are involved in up-regulation of MC receptor genes. Decreased expression of the analyzed genes in the lesional skin is not surprising, but statistically significant increased
* Corresponding author at: Department of Physiology, University of Tartu, 19 Ravila Street, 50411 Tartu, Estonia. Tel.: +372 7 374 335; fax: +372 7 374 332. E-mail address: [email protected] (S. Ko ˜ks). 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.06.004
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K. Kingo et al. expression of studied genes in non-lesional skin from vitiligo patients is not described previously. Conclusion: In our mind, up-regulation of melanocortin system in non-lesional skin could be systemic compensation to restore normal pigmentation in lesions. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Vitiligo is an acquired cutaneous disorder that presents with gradual skin depigmentation produced by the deterioration of melanocyte functions. Several hypotheses have proposed to explain the dysfunction and/or loss of melanocytes in epidermis of vitiligo patients [1,2]. These include an autoimmune mechanism, an auto-cytotoxic mechanism and an abnormality in melanocytes or in surrounding keratinocytes-producing factors necessary for the survival and function of melanocytes [3—5]. Till now the pathogenesis of vitiligo remains partially understood and probably involves various combinations of diverse mechanisms. The melanocortin system is an evolutionarily conserved regulatory module, operating as a coordinator and executor of responses to stress. The melanocortin system consists of the melanocortin peptides a-, b- and g-melanocyte-stimulating hormone (a-MSH, b-MSH and g-MSH) and adrenocorticotropic hormone (ACTH), that are post-translational products of the proopiomelanocortin (POMC) prohormone gene; the endogenous melanocortin receptor antagonists: agouti (ASIP) and agouti-related protein (AGRP); the family of five melanocortin receptors (MCRs) that exert the biological effects of the POMC peptides [6]. The components of the melanocortin system and their relationships are shown in Fig. 1. Described system is mainly expressed in the brain but also in many peripheral tissues including the skin. In the skin, the components of the melanocortin system are expressed in different cells. POMC mRNA is detected in the keratinocytes and melanocytes of normal epidermis and in the pilosebaceous unit [7]. Among POMC-derived peptides a-MSH and ACTH are the most abundant in the skin [8,9]. MC1R, the most strongly expressed melanocortin receptor in the human skin, has its highest expression in melanocytes [10]. The most active peptide for MC1R is a-MSH followed by ACTH, while b-MSH and g-MSH cause weak activation of this receptor [11]. The expression of MC2R has been detected in the melanocytes and adipocytes and this receptor is activated by ACTH [11,12]. MC4R, which is activated both by a-MSH and ACTH, has been reported to be expressed in dermal papilla cells [11,13]. The expression of MC5R has been established in sebocytes, adipocytes and skin mast
cells [12—14]. The binding of the POMC peptides to this receptor is similar to that of MC1R. No manifestation of MC3R mRNA in human skin has been reported. Functional purpose of the melanocortin system in the skin is to respond to external and internal stresses through local pigmentation, immune, epidermal, adnexal and vascular structures to stabilize skin function and prevent disruption of internal homeostasis [15,16]. Components of the melanocortin system are involved in determining skin and hair phenotypes as well as in different skin inflammatory disorders and malignancies. An association between the variability of the POMC gene and red hair phenotype has been demonstrated [17]. The abnormal expression of POMC peptides has been established in severe atopic dermatitis, psoriasis, scarring alopecia and inflammatory keloids as well as in the nodular type and metastatic melanomas and basal cell carcinoma [16]. The MC1R gene variants are linked with red hair and poor tanning ability [18—21]. Additionally, MC1R has been reported as a genetic
Fig. 1 The components of the melanocortin system: POMC, proopiomelanocortin; ASIP, agouti signalling protein; a-MSH, alpha-melanocyte-stimulating hormone; bMSH, beta-melanocyte-stimulating hormone; g-MSH, gamma-melanocyte-stimulating hormone; ACTH, adrenocorticotropic hormone; AGRP, agouti-related protein; MC1R, melanocortin receptor 1; MC2R, melanocortin receptor 2; MC3R, melanocortin receptor 3; MC4R, melanocortin receptor 4; MC5R, melanocortin receptor 5.
Melanocortin system expression in vitiligo
115 been studied, but association between variations of these genes and susceptibility to vitiligo has not been proved [30]. No studies have analyzed gene expression levels of the melanocortin system in vitiligo samples up to the present time.
2. Results Fig. 2 MC1R, MC2R, MC3R, MC4R and MC5R mRNA levels (relative to housekeeping gene HPRT mRNA level) in the skin from healthy controls (HCS).
risk factor for melanoma, basal cell carcinoma and squamous cell carcinoma [22,17]. ASIP inhibits the binding of a-MSH to MC1R, with resulting inhibition of melanogenesis [23,24]. Polymorphism in the ASIP gene at position 8818 was found to be associated with dark hair and brown eyes [25]. The role of AGRP in pigmentation has not been verified. As AGRP blocks a-MSH binding to melanocortin receptors, it is possibly implicated in human pigmentation [26]. The purpose of present study was to examine the expression variations of genes encoding mediators of the melanocortin system in non-lesional and lesional skin of vitiligo patients and in skin of healthy controls with the aim to explore the regulation of the cutaneous stress response system in vitiligo. In previous studies, a reduction in the level of the POMC peptide a-MSH has been demonstrated both in lesional skin and serum of vitiligo patients [27,28]. Moreover, Graham et al. demonstrated using a-MSH immunoreactive positive melanocytes that low expression of a-MSH in the lesional skin of vitiligo patients resulted from decreased expression of the peptide rather than a reduction in melanocyte numbers [29]. The relationship between vitiligo and MC1R and ASIP gene polymorphisms has also
Fig. 3 POMC mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. *p < 0.05.
Gene expression levels of POMC, the five melanocortin receptors (MC1R—MC5R) and endogenous melanocortin receptor antagonists (ASIP and AGRP) were measured by quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) in punch biopsies from lesional and non-lesional skin of vitiligo patients (n = 31) and from non-sun-exposed skin of healthy subjects (n = 24). In addition, levels of two genes encoding enzymes concerned with melanogenesis — tyrosinase-related protein-1 (TRP1) and dopachrome tautomerase (DCT) — were measured. The presence of mRNA expression of examined genes in skin of healthy controls and vitiligo patients became evident in QRT-PCR with a relatively large number (27—36) of amplification cycles. In the samples, MC1R demonstrated the highest expression (amplification after 27 cycles), whereas the levels of MC2R, MC3R, MC4R and MC5R mRNAs were low (amplification after 32—36 cycles) (Fig. 2). POMC mRNA was detected at 28—32 cycles, ASIP and AGRP at 34—36 cycles. TRP1 and DCT mRNAs were detected at 28—30 cycles. None of the studied genes had statistically significant gender-related differences in mRNA expression levels. A difference in POMC mRNA expression level, although marginally satisfying the standard criteria of statistical significance ( p < 0.05), was evident between lesional and non-lesional skin of vitiligo subjects (Fig. 3). The POMC mRNA expression was 1.9 fold lower in involved skin compared with uninvolved skin in the vitiligo group. POMC expression was similar in uninvolved vitiligo skin and in skin biopsies of healthy controls. No significant differences in ASIP expression as well as AGRP expression were established when comparing the study groups (data not shown). Statistically significant differences in MC1R expression and MC4R expression between healthy controls and vitiligo patients were established. Specifically, MC1R mRNA expression was 1.6 fold higher in non-lesional skin of vitiligo patients when compared to healthy subjects ( p < 0.01; Fig. 4(a)). Likewise, the MC4R expression level in vitiligo non-lesional skin was 1.9 fold higher than in healthy skin ( p < 0.01; Fig. 4(b)). In lesional skin, 2.1 fold decrease of MC1R ( p < 0.0001; Fig. 4(a)) and 2.5 fold decrease of MC4R ( p < 0.01; Fig. 4(b)) expressions were detected
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Fig. 4 MC1R (a) and MC4R (b) mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. **p < 0.01 compared to healthy control; ++p < 0.01, +++p < 0.0001 compared to non-lesional skin.
when compared to non-lesional skin samples from patients with vitiligo. The mRNA expression levels of the three other melanocortin receptors (MC2R, MC3R and MC5R) were also increased in unaffected skin and decreased in lesional skin of vitiligo patients; however, the differences were not statistically significant (data not shown). Suppression of TRP1 and DCT expressions in lesional skin compared to non-lesional vitiligo skin and healthy controls was established. Specifically, 6.8 fold decrease of TRP1 mRNA expression in involved skin compared with skin of healthy subjects ( p < 0.0001; Fig. 5(a)) and 19.7 fold decrease of TRP1 in lesional skin compared to uninvolved skin in the vitiligo group ( p < 0.0001; Fig. 5(a)) was detected. The DCT mRNA expression was 7.6 fold lower in involved skin compared with skin of healthy controls ( p < 0.0001; Fig. 5(b)) and 12.9 fold lower in lesional skin compared to uninvolved skin in the vitiligo group ( p < 0.0001; Fig. 5(b)). Contrarily, the TRP1 mRNA expression was 2.9 fold higher in nonlesional skin of vitiligo patients compared to healthy controls ( p < 0.05; Fig. 5(a)). The DCT expression level in non-lesional skin of vitiligo patients showed
K. Kingo et al.
Fig. 5 TRP1 (a) and DCT (b) mRNA levels (relative to housekeeping gene HPRT mRNA level) in skin from healthy controls (HCS), non-lesional vitiligo skin (NLS) and lesional vitiligo skin (LS). Bars indicate mean S.E.M. *p < 0.05, *** p < 0.0001 compared to healthy control; +++p < 0.0001 compared to non-lesional skin.
also a clear tendency of increase when compared to healthy subjects while not reaching the level of statistical significance ( p = 0.14). No differences in the expressions of genes of the melanocortin system and genes involved in melanin synthesis were detected between subgroups of vitiligo based on the extent of involvement and progression of the disease. Possible interactions between the expressions of the studied genes were examined by Spearman rank correlation. In addition, the correlations between the expressions of genes of the melanocortin system and tyrosinase (TYR), essential enzyme in melanin synthesis, were examined. In our previous study, a statistically significant decrease in TYR mRNA expression was observed in lesional skin compared to non-lesional skin of vitiligo patients and in skin of healthy subjects (both p < 0.0001) [31]. TYR expression was elevated in uninvolved vitiligo skin compared to skin samples from controls, but this difference was not statistically significant. The data of the correlations between the expressions of the genes of the melanocortin system in the control group and patient group are presented in Table 1.
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0.57 /0.22/0.27 0.29/0.05/0.17 0.21/0.11/0.29 0.01/0.15/0.45 * 0.60*/0.54**/0.60 ** 0.79***/0.79***/0.83 *** 1/1/1 0.36/0.51**/0.62 *** 0.54 /0.28/0.24 0.11/0.15/0.36 0.29/0.27/0.16 0.02/0.09/0.17 0.60*/0.63***/0.44 * 1/1/1 0.79***/0.79***/0.82 *** 0.37/0.26/0.76 *** 0.07/0.31/ 0.28/0.052/0.23 0.27/0.02/0.24 0.43/0.29/0.30 1/1/1 0.60*/0.63***/0.44 * 0.60*/0.54**/0.60 ** 0.41/0.20/0.28 0.1/0.07/0.15 0.73***/0.27/0.17 0.06/0.09/0.36 1/1/1 0.43/0.29/0.30 0.02/0.09/0.23 0.01/0.15/0.45 * 0.13/0.21/0.24 0.05/0.14/0.03 0.01/0.04/0.12 1/1/1 0.06/0.09/0.36 0.27/0.02/0.24 0.29/0.27/0.16 0.21/0.11/0.29 0.35/0.28/0.01
0.32/0.42 /0.16 1/1/1 0.01/0.04/0.12 0.73***/0.27/0.17 0.28/0.05/0.23 0.11/0.15/0.36 0.29/0.05/0.17 0.21/0.35/0.29 p < 0.05. p < 0.01. p < 0.001. ***
*
**
1/1/1 0.32/0.42*/0.16 0.05/0.14/0.02 0.11/0.07/0.15 0.07/0.31/0.29 0.54*/0.28/0.24 0.57**/0.22/0.27 0.41/0.12/0.26 POMC ASIP AGRP MC1R MC2R MC3R MC4R MC5R
MC5R (HCS/NLS/LS) MC4R (HCS/NLS/LS)
** *
MC3R (HCS/NLS/LS) MC2R (HCS/NLS/LS) MC1R (HCS/NLS/LS) AGRP (HCS/NLS/LS) ASIP (HCS/NLS/LS) POMC (HCS/NLS/LS)
Thehypothalamic—pituitary—adrenalaxis(HPA)inthe brain is the main mediator of the systemic response to stress.TheskinexpressesanequivalentoftheHPAaxis: a cutaneous defence system that operates as a coordinator and executor of local responses to stress. The melanocortin system in the skin is a part of the cutaneous HPA axis [12]. In the present study, we analyzed expression of melanocortin system genes in skin samples from patients with vitiligo. Our study sample contains 25 patients with generalized vitiligo, 1 with segmental vitiligo, 4 with local form and 1 with universal form. As segmental vitiligo and generalized vitiligomayhavedifferentpathogenesis,wecompared meanexpressionsofstudiedgenesindifferentformsof vitiligo. There was no significant difference in the expression pattern and we decided to merge the expression results of different forms (segmental, generalized, localized and universal) of vitiligo into one study group. Genes of the melanocortin system analyzed in the human skin in the present study were proopiomelanocortin (POMC), five melanocortin receptors (MC1R—MC5R), agouti signalling protein (ASIP) and agouti-related protein (AGRP). Our results reaffirmed the expression of cutaneous stress response system in human skin, whereas expression of MC3R mRNA in human skin has not previously been reported. Further studies using histochemical methods must be conducted to localize MC3R expression to distinct cell types in the skin. The expression profiles of all five melanocortin receptors were analogous between the study groups, a markedly significant difference between lesional and nonlesional vitiligo skin ( p < 0.0001) was established only in the case of MC1R. Probably, the expression levels of the genes indicate their functional relevance. In our samples, MC1R demonstrated the highest expression, while the rest of the melanocortin
Table 1 Spearman rank correlation coefficient (Spearman r) for expression values of melanocortin system
3. Discussion
*
Variables of the mRNA expression of different components of the melanocortin system were positively related. Among the expressions of TYR, TRP1 and DCT mRNAs, a strong positive correlation equally in skin of healthy controls and in skin of vitiligo patients was observed (r > 0.70; p < 0.0001; Fig. 6(a—c)). Further, positive correlation was noted between the levels of MC1R and TRP1 in skin of healthy controls (r = 0.47; 95% CI 0.001—0.77; p < 0.05; Fig. 7(a)). Contrarily, no statistically significant correlation between variables of MC1R mRNA and expressions of TYR, TRP1 and DCT mRNAs were observed in non-lesional and lesional vitiligo skin (Fig. 7(b and c)).
0.41/0.12/0.26 0.21/0.35/0.29 0.35/0.28/0.01 0.13/0.21/0.24 0.41/0.20/0.28 0.37/0.26/0.76 *** 0.36/0.51**/0.62 *** 1/1/1
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K. Kingo et al.
Fig. 6 (a) The relation of the levels of TYR (TYR_HCS), TRP1 (TRP1_HCS) and DCT (DCT_HCS) in skin of healthy subjects. (b) The relation of the levels of TYR (TYR_NLS), TRP1 (TRP1_NLS) and DCT (DCT_NLS) in non-lesional vitiligo skin. (c) The relation of the levels of TYR (TYR_LS), TRP1 (TRP1_LS) and DCT (DCT_LS) in lesional vitiligo skin.
receptors had relatively low expression levels. Moreover, the correlation analysis presented a moderate up to strong positive relation between the levels of mRNA of the different melanocortin receptors. High homology in sequences may be the cause of the observed similar expression profile of the melanocortin receptors [11]. Alternatively, this kind of expression profile of the melanocortin receptors could be explained by concomitant expression of these genes. Concomitant expression of MC1R and MC2R has been reported in human keratinocytes and epidermal melanocytes [32—34]. Concomitant expression of MC1R and MC4R has been observed in human dermal papilla cells [35]. In the skin, the potential role of the melanocortin system in the network of cutaneous inflammation and pigmentation has been verified. The possible role of markers of the melanocortin system in the pathogenesis of vitiligo remains unclear. In the present study, POMC mRNA expression was found to be lower in lesional skin compared with non-lesional
skin in the vitiligo group ( p < 0.05). At the same time, we did not detect difference in the expression of POMC between non-lesional skin from vitiligo patients and skin from healthy controls. The mRNA expression levels of the melanocortin receptors, mediating the effects of POMC peptides, were decreased in lesional skin compared to uninvolved skin and increased in unaffected skin of vitiligo patients compared to healthy subjects, the differences were statistically significant ( p < 0.01) in the cases of MC1R and MC4R. Decreased expression of MC1R in lesional skin is not surprising, because this fits the loss of functional melanocytes–—major cell type expressing MC1R. Contrarily, MC4R is expressed only in dermal papilla cells. Similar down-regulation of MC1R and MC4R in lesional skin may occur due to high homology in their sequences, which may provide similar regulatory regions employed in lesional skin. Increased expression of MC receptors in nonlesional skin of patients is not so well understood. This is not caused by natural UVR exposure, because
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Fig. 7 (a) The relation of the levels of MC1R (MC1R_HCS) and TYR (TYR_HCS), TRP1 (TRP1_HCS) and DCT (DCT_HCS) in skin of healthy controls. (b) The relation of the levels of MC1R (MC1R_NLS) and TYR (TYR_NLS), TRP1 (TRP1_NLS) and DCT (DCT_NLS) in non-lesional vitiligo skin. (c) The relation of the levels of MC1R (MC1R_LS) and TYR (TYR_LS), TRP1 (TRP1_LS) and DCT (DCT_LS) in lesional vitiligo skin.
skin biopsies were taken from truly non-exposed areas. Also, distribution of skin phototypes between study groups was balanced. Over-expression of MC receptors possibly indicates the existence of compensation system to restore normal pigmentation in lesions. Lack of difference in case of POMC in the non-lesional and control skin samples suggest that this over-expression is induced by systemic, circulating influences rather than local ones. Tyrosinase (TYR) and tyrosinase-related proteins (TRP1 and DCT) are closely related melanocytespecific gene products involved in melanogenesis and in the biology of melanocytes. Down-regulation of TYR mRNA expression has been documented in lesional skin of vitiligo patients [36]. Accordingly, in our previous study, a statistically significant decrease in TYR expression was observed in lesional skin compared to non-lesional skin of vitiligo patients and healthy control subjects (both p < 0.0001) [31]. In addition, significantly lower level of TRP1 mRNA expression by vitiligo melanocytes compared to control normal melanocytes is established [37]. No expression of DCT mRNA has
been investigated in vitiligo up to the present time. In the present study, suppression of expressions of TRP1 and DCT mRNAs, as verification of decreased melanin synthesis in vitiligo, was established in lesional skin of vitiligo patients. Current literature indeed suggests that the lesional area is characterized by the absence of melanocytes or by the presence of ineffective melanocytes. However, not all melanocytes are destroyed in lesions and this explains why these mRNAs are still in detectable level. We found expressional up-regulation of genes involved in melanin synthesis in uninvolved skin samples of vitiligo patients compared to healthy control samples. In case of TRP1, this difference was statistically significant. As we also found overexpression of melanocortin receptors in the same samples, activation of enzyme genes in non-lesional skin is a consequence of melanocortin stimulation. This indicates functional relevance of our finding. It is possible that the suppression of melanin production activates melanocortin receptors transcriptionally through negative feedback to restore normal
120 pigmentation. On the other hand, melanocortins have the widest spectrum of immunomodulatory/ anti-inflammatory capacities [38—41]. Increased expressions of pro-inflammatory cytokines (IL-2, TNF-a, IL-6 and INF-g) in lesional skin compared to uninvolved skin of vitiligo patients have been reported [42,43]. Up-regulation of the melanocortin receptors in non-lesional skin, established in the present study, may attempt to inhibit the production or action of proinflammatory factors and thereby make an effort to suppress inflammatory responses in lesional skin of vitiligo patients. Positive correlation between the levels of MC1R and TRP1 was found in the skin of healthy controls, this correlation did not exist in patients. Described finding indicates that significant changes in transcriptional regulation of the melanocortin and melanin synthesis system occur in skin during vitiligo. In conclusion, the expression of genes of the melanocortin system is altered in vitiligo. Decreased expression of the genes of the melanocortin system and enzymes of melanogenesis in the lesional skin demonstrated in the present study is not surprising and fits with existing data. On the other hand, statistically significant increased expression in non-lesional skin from vitiligo patients is not described previously. In our mind, this up-regulation could be systemic compensation to restore normal pigmentation in lesions.
4. Materials and methods The study protocols and informed consent forms were approved by the Ethical Review Committee on Human Research of the University of Tartu. All participants signed a written informed consent. The patients and control subjects in the study were Caucasians living in Estonia. Unrelated patients with vitiligo (n = 31; 22 females; 9 males; age range 22—75 years) from the Department of Dermatology, the University of Tartu, were included in the study. The mean age of vitiligo onset of the patients was 30.0 years and the mean duration of vitiligo was 19.2 years. Five patients had a family history of vitiligo. None of the patients included in the study had received specific therapy in the previous 6 months. The clinical signs on which the diagnosis of vitiligo was based on were characteristic loss of skin pigmentation with typical localization and white colour on the skin lesions under Woods lamp. The type of vitiligo was based on the extent of involvement and the distribution of pigmentation. Focal vitiligo involves depigmentation in a localized, non-dermatomal distribution (n = 4(F)). Segmental vitiligo encompasses depigmentation of the dermatomal,
K. Kingo et al. asymmetric distribution (n = 1(F)). Generalized vitiligo is characterized by bilateral, symmetric loss of pigmentation of the torso, face, neck, or extensor surfaces of the hands and legs (n = 25; 17(F); 8(M)). Universal vitiligo occurs as depigmentation of the entire body surface area (n = 1(M)). The stage of vitiligo was based on the interval of manifestation of new areas of depigmentation or enlargement of the area of depigmentation. The patients were divided into two subgroups based on the stage of progression of the disorder: patients with progressive vitiligo (active vitiligo, in which new areas of depigmentation or enlargement of depigmentation were observed during the previous 3 months; n = 22; 16(F); 6(M)) and patients with stable vitiligo (inactive vitiligo, in which no new depigmentation or enlargement of depigmentation had been observed for more than 3 months; n = 9; 6(F); 3(M)). The control group consisted of healthy volunteers (n = 24; 17 female; 7 male; age range 21—67 years) free from the positive family history of vitiligo and other chronic dermatoses. Control subjects were recruited from health care personnel, medical students and patients present at the dermatological outpatient clinic with either facial teleangiectasis or skin tags. Two skin biopsies (Ø 4 mm) were obtained from each patient with vitiligo: one from the central part of involved skin and another from non-sun-exposed uninvolved skin. One skin biopsy (Ø 4 mm) from nonsun-exposed skin was taken from healthy control subjects. The non-sun-exposed skin was defined as the skin never exposed to UVR previously and definitely not exposed to natural UVR in the last 12 months. Biopsies from uninvolved skin of vitiligo patients and healthy controls were taken from the lower abdomen. All probands had skin phototype II (9 controls, 13 patients) or III (15 controls, 18 patients), Fitzpatrick classification [44]. Biopsies were instantaneously snap-frozen in liquid nitrogen and stored at 80 8C until further use. Total RNA was isolated from tissues using RNeasy Fibrous Tissue Mini Kit (QIAGEN Sciences, MD, USA) following the protocol suggested by the manufacturer. For tissue homogenization, Ultra-Turrax T8 homogenizer (IKA Labortechnik, Germany) was used. RNA was dissolved in RNase free water and stored until further use at 80 8C. For each RT-PCR reaction, 500 ng of total RNA was converted into cDNA. The reverse transcription reactions were performed with a reverse transcriptase (SuperScript III; Invitrogen Corp., Carlsbad, CA, USA) and poly(T18) oligonucleotides in accordance with the manufacturer’s instruction. The reaction mixtures were incubated at 65 8C for 5 min, at 0 8C for 1 min, at 50 8C for 90 min, at 70 8C for 5 min and finally stored at 80 8C.
Melanocortin system expression in vitiligo Gene expression levels were detected in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Reactions were carried out in 10 ml reaction volumes in four replicates. The expression levels of AGRP, ASIP, the melanocortin receptor genes and TRP1 and DCT were detected applying TaqMan-QRT-PCR method using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). For the detection of the expression levels of AGRP, ASIP, MC3R, MC4R, MC5R, TRP1 and DCT, we used TaqMan Assay-OnDemand FAM-labelled MGB-probe gene expression assay mixes (20X, Applied Biosystems, Foster City, CA, USA). The assay mixes used were Hs00361403_g1 (AGRP), Hs00181770_m1 (ASIP), Hs00252036_s1 (MC3R), Hs00271877_s1 (MC4R), Hs00271882_s1 (MC5R), Hs00167051_m1 (TRP1) and Hs00157244_ m1 (DCT). For the detection of the expression levels of MC1R and MC2R, we used gene-specific primers (MC1R: forward 50 -TGCGGCTGCATCTTCAAG-30 , reverse 50 -TGATGGCATTGCAGATGATGA-30 ; MC2R: forward 50 -CTCGATCCCACACCAGGAA-30 , reverse 50 TGTGATGGCCCCTTTCATGT-30 ) and MGB-labelled probe (MC1R: FAM-TTCAACCTCTTTCTCGCC-NFQ; MC2R: FAM-TCTCCACCCTCCCCAGA-NFQ). For the detection of HPRT-1 (hypoxanthine phosphoribosyltransferase-1) expression level, gene-specific primers (HPRT-1 exon 6, 50 -GACTTTGCTTTCCTTGGTCAGG-30 ; HPRT-1 exon 7, 50 -AGTCTGGCTTATATCCAACACTTCG-30 ; final concentrations 300 nM) and VIC-TAMRA-labelled probe (VIC-50 -TTTCACCAGCAAGCTTGCGACCTTGA-30 -TAMRA; final concentration 200 nM) were used. The expression level of POMC was detected using qPCR Core Kit for SYBR Green I (Eurogentec, Seraing, Belgium) and gene-specific primers (forward 50 CTACGGCGGTTTCATGACCT-30 , reverse 50 -CCCTCACTCGCCCTTCTTG-30 , final concentrations 100 nM). For quantification of mRNA, we used comparative Ct method (DCt value), where the amount of target transcript was normalized according to the level of endogenous reference HPRT-1 (hypoxanthine phosphoribosyl-transferase-1). Adjustment to normal distribution was tested by the Kolmogorov—Smirnov test. The distribution of measurements of gene expressions by the applied method did not follow a Gaussian distribution. Mann—Whitney U-test and Kruskal—Wallis test were used to test for differences between the groups using GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA). Correlation analysis was used to investigate relations between two parameters of one group. For measure of correlation, the Spearman rank correlation was applied. For all tests, a p value < 0.05 was considered significant.
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Acknowledgements This study was financially supported by the targetbased funding from the Estonian Ministry of Education Grant No. SF0180043s07, by University of Tartu Research Grant PARFS 05901, by the Estonian Science Foundation Grants No. 6576, 5712 and 5688 and by the Centre of Molecular and Clinical Medicine Grant VARMC-TIPP.
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