ABSTARCT

The nuclear receptor LXR-α is a transcriptional regulator involved in numerousepidermal processes including proliferation, differentiation, permeability barrierformation, inflammatory responses, skin development and homeostasis. Owing to itscrucial for multiple cell types in the skin, its activation in one skin cell type mayinfluence its expression and activation in other, thereby having a functional impact. Inthis study we investigated the effects that LXR-α activation in keratinocytes would exerton LXR-α expression in melanocytes. For this, we cultured melanocytes from theclinically healthy subjects and them nurtured with the media from the LXR-α activated (by both Ascorbic acid and Atorvastatin along with 22-R hydroxycholestrol) keratinocyte. The DOPA staining verified the growth of melanocytes and the validationfor viability was done by flow cytometry. The results so obtained supported ourspeculation that LXR-α activation in the normal healthy melanocytes may lead to theirapoptosis. Therefore, LXR-α may be a critical player in keratinocyte and melanocytebiology and could be a potential target for skin disease management.

INTRODUCTION

Epidermal melanocytes form a functional and structural unit with neighboring keratinocyte. There is apparently a close relationship between melanocytes and keratinocytes that is important for melanocyte survival and differentiation. and that may involve keratinocyte-mediated cytokines [1]. Growth factors produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes [2-5]. Therefore, changes in keratinocytes function might have a significant effect on melanocyte survival [6, 7]. The LXRs in skin physiology and pathology have evolved rapidly in recent years as they modulate epidermal proliferation, carcinogenesis, differentiation and permeability barrier function, which identifies them as promising drug targets for the treatment of skin diseases. The nuclear receptors LXR-α and LXR-β are expressed in murine and human keratinocytes [8, 9]. LXR activation also stimulates epidermal lipid synthesis, lamellar body secretion and lipid processing in the stratum corneum [10]. LXR-αactivators stimulate keratinocyte differentiation and also promote epidermal permeability barrier homoeostasis [10]. Activation of LXR-αby oxysterols stimulates keratinocyte differentiation, thereby, making LXR-αimportant in keratinocytes differentiation as well [11, 12]. LXR-αis also known to play a key role as metabolic checkpoint that modulates cell proliferation in skin. At proper dosage, synthetic LXR agonists are safe on endothelial cells and may even transrepress inflammatory reactions [13].It has also been found that LXR-α might be playing an important role in pathogenesis of pigmentary disorders like psoriasis [14, 15]and vitiligo [16]. Changes in the expression of this receptor in various diseased conditions of skin make it a candidate gene worth investigation, as it may be critical players in keratinocyte and melanocyte biology and homeostasis [17].

In this article we characterize the effect of alteration in expression of LXR-α in the keratinocytes influence the survival of the melanocytes. In our previous studies we have already explored the effects of agonists and activators of LXR-α on its own gene expression in keratinocytes. We here report the effect of melanocytes viability following LXR-α activation with Atorvastatin+22R hydroxycholestrol and Ascorbic acid +22R hydroxycholestrolin cultured keratinocytes, with both the cell types derived from of the same the skin biopsy

METHODS

Selection of the subjects and clinical evaluation

This study was approved by the Institutional Ethics Committee. A total number of 6 controls were enrolled, after their informed consent. The age range was 18–40 years. Skin grafts were collected in the phosphate buffer saline (PBS) and immediately transported to the laboratory in ice.

Cellular models employed

Fresh biopsy specimens were obtained under aseptic conditions in phosphate buffer saline with antibiotics (penicillin and streptomycin).

1. Keratinocyte Cultures: Culturing of keratinocytes derived from skin biopsies of clinically healthy subjects were carried out in Keratinocytes Specific Media containing no antibiotics. The treatment with Atorvastatin+22R hydroxycholestrol and Ascorbic acid +22R hydroxycholestrol was performed. Cells in one of the wells were incubated with 30µM Atorvastatin and the other well was treated with 0.2mg/ml Ascorbic acid or 12 hours [18]. Then 10 µM 22R hydroxycholestrol was added to both the wells and cells were then incubated for 48 hours.
2. Melanocyte Cultures: Culturing of melanocytes derived from skin biopsies of same clinically healthy subjects was carried out in Melanocyte Media Promocell containing no antibiotics. Then the media from the above mentioned treated keratinocytes was transferred to the respective melanocytes cultures for consecutive three days.

Cell identification

1. Keratinocytes: To verify that the cells cultured from the skin biopsies exhibited the characteristic signatures of keratinocytes,
2. Melanocytes :DOPA staining To verify that the cells cultured from the skin biopsies exhibited the characteristic signatures of melanocytes, DOPA staining was performed following a modified method previously described [19].

RNA isolation and cDNA synthesis

Total RNA was isolated using the Tri Reagent kit (Ambion, Austin, TX, USA), and cDNA was synthesized using the First-Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany) following the manufacturers’ protocols.

Semiquantitative RT-PCR

Semiquantitative RT-PCR was used to determine the gene tran- scriptional expression. PCR amplification was performed using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). All primers were synthesized by Sigma (St. Louis, MO, USA). The primer sequences used are given in Table S2. PCR amplification of cDNA was performed in a reaction mixture containing 10X polymerase, 2 ll cDNA template and sterile RNAse-free water added to a total volume of 25 ll. All PCR reagents were from Fermentas. We first amplified a housekeeping gene encoding b-actin, to monitor RNA quality and cDNA synthesis and to ensure that equivalent amounts of cDNA were used in all PCR amplifications. All PCR products were analysed by separation on a 2% agarose gel stained with ethidium bromide.

Annexin V staining

Cultured melanocytes after culturing in conditioned media were were processed as previously mentioned [20] before being used for Annexin V staining (Roche, Mannheim, Germany), according to the manufacturer’s instructions.

RESULTS

Identifications of melanocytes

Melanocytes were cultured with conditioned media from treated keratinocytes (both cell types derived from from skin biopsies of the same patient). After getting pure cultures, these cells were characterized by DOPA staining (Figure 1).

LXR-α mRNA expression

We checked the expression profile of LXR-α gene in melanocytes cultured in the conditioned media was compared to the controls (Figure.2). The aim was to detect any change in gene expression of LXR-α and its effector genes . Results revealed the higher presence of LXR-α mRNA expression in melanocytes cultured in bot the treated conditioned media compared to controls.

Effect on the apoptosis

Experiments were performed and it was interesting to find that there was an increase in the apoptosis of melanocytes nurtured with the media transferred from the keratinocytes treated Ascorbic acid + 22-R hydroxycholestrol i.e. 26% compared to 17.6% in the melanocytes nurtured with the media transferred from the keratinocytes treated Atorvastatin + 22-R hydroxycholestrol whereas the control non- treated melanocytes showed 10% apoptotic cell population (Figure 3).

DISCUSSION

The role multivalent LXR-α has recently been described in many skin diseases. A marked expression of LXR-α has been observed in cells adjacent to dermal papilla, speculating that it may correlate with site of hair melanocytes [21]. Important genes involved in regulation of both keratinocytes and melanocytes are target genes of LXR-α; it can be speculated that LXR-α might be playing the important role in pathogenesis of varied skin disorders and homeostasis [17].

Studies have previously shown that chronic activation of LXR-α in pancreatic β-cell provoked lipid dysregulation and concomitant apoptosis. To verify the speculation, the cultured melanocytes from the clinically healthy subjects were nurtured with the media from the LXR-α activated (by both Vitamin C and Atorvastatin alongwith 22-R hydroxycholestrol) keratinocyte media. The DOPA staining in Figure 1 shows the viable melanocytes which were further validated by FACS and the results so obtained supported our speculation that LXR-α activation in the normal healthy melanocytes may lead to their apoptosis, as LXR-α is known to inhibit cell proliferation and enhance apoptosis (Figure 3). We have already reported that the LXR-α expression was present in human melanocytes and keratinocytes [15, 16]. In this study, we compared the expression of LXR-α in conditioned media from keratinocytes treated with Ascorbic acid + 22-R hydroxycholestrol and Atorvastatin + 22-R hydroxycholestrol compared to the control and found that mRNA expression of LXR-α was significantly higher in both the treated groups as compared to the control.So, it can be said that there is an LXR-α imbalance in the genesis of skin disorders. Although future studies will reveal whether LXR-α dysregulation in skin cells contributes to the diseased state in vivo, the data presented here suggest a potential target for the development of a successful method of regulating the diseased skin conditions. Not only LXR-α has a robust anti-inflammatory activity in skin, but they also modulate epidermal proliferation, differentiation and permeability barrier function. The abnormal increase in LXR-αexpression in the pancreatic islets of obese and diabetic animal models and the ability of LXR-αligands to induce cell dysfunction suggest the involvement of chronic LXR-αin cell apoptosis [22] . Keeping in view, the findings reported here coupled with earlier reported findings, it is not unlikely that LXR-α transcriptome may be of crucial importance, not only in understanding of genomic basis of skin disorders it could be useful in designing futuristic therapy for these skin disorders.