Abnormal expression of the vitamin D receptor in keloid scars
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], [3], [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 20-times 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], [8], [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-β1) pathway. TGF-β1, 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-β1 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-β1 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-β1 [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-β1-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], [15], [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], [18], [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], [21], [22]. Hypothetically, reduced vitamin D levels after burn may also contribute to the risk of abnormal scar formation, which may be further 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 ligand-bound 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 half-life, 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.
Section snippets
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,
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. 17.5 ± 12.1 years, respectively; p < 0.001). There was also a statistically significant difference in donor gender between the
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
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.
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