Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to α-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor

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Abstract

Nitric oxide (NO) and α-melanocyte-stimulating hormone (α-MSH) have been correlated with the synthesis of melanin. The NO-dependent signaling of cellular response to activate the hypothalamopituitary proopiomelanocortin system, thereby enhances the hypophysial secretion of α-MSH to stimulate α-MSH-receptor responsive cells. In this study we investigated whether an NO-induced pathway can enhance the ability of the melanocyte to respond to α-MSH on melanogenesis in alpaca skin melanocytes in vitro. It is important for us to know how to enhance the coat color of alpaca. We set up three groups for experiments using the third passage number of alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures like the control group but the melanocytes were then irradiated everyday (once) with 312 mJ/cm2 of UVB; the UV + L-NAME group is the same as group UV but has the addition of 300 μM L-NAME (every 6 h). To determine the inhibited effect of NO produce, NO produces were measured. To determine the effect of the NO to the key protein and gene of α-MSH pathway on melanogenesis, the key gene and protein of the α-MSH pathway were measured by quantitative real-time PCR and Western immunoblotting. The results provide exciting new evidence that NO can enhance α-MSH pathway in alpaca skin melanocytes by elevated MC1R. And we suggest that the NO pathway may more rapidly cause the synthesis of melanin in alpaca skin under UV, which at that time elevates the expression of MC1R and stimulates the keratinocytes to secrete α-MSH to enhance the α-MSH pathway on melanogenesis. This process will be of considerable interest in future studies.

Introduction

Ultraviolet (UV) B radiation can cause skin-tanning via the synthesis of melanin which is synthesized by specific tyrosinase and tyrosinase-related enzymes expressed in melanocytes. It is reported that several melanogenic factors are released from keratinocytes and other cells surrounding melanocytes in the skin following UV radiation. These melanogenic factors include nitric oxide (NO) [1], adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH) [2], [3], endothelin-1 (ET-1) [4], [5], prostaglandins [6], thymidine dinucleotide [7], and histamine [8]. Some of them are reported to up-regulate tyrosinase gene expression through different pathways.

NO is considered to play an important role in UV-induced hyperpigmentation [1], [9]. It is generated from l-arginine by the catalytic action of three NO synthase (NOS) isoforms (neuronal nNOS or NOS1, inducible iNOS or NOS2, and endothelial eNOS or NOS3) [10], [11], [12]. The downstream targets of NO in melanocytes remain to be fully elucidated. It is likely that NO plays a central role in the activation of tyrosinase via guanylate cyclase and PKG activation [1]. It may also be involved in the induction of the tyrosinase mRNA message, which is induced within 2 h of an application invitro of an NO donor [9]. Other NO-activated pathways, however, might also be important. In the presence of oxygen, NO reacts with the melanin related metabolites 5,6-dihydroxyindole and its 2-carboxylic acid (DHICA) resulting in the deposition of melanin-like pigments [13]. Evidence from other cell systems suggests that NO can reduce inositol triphosphate (IP3) synthesis [14] and reduce the release of Ca2+ via modulation of the IP3 receptor [15], [16]. The enhancement of tyrosinase gene expression via the cyclic guanosine 3′,5′-monophosphate (cGMP) pathway may be a primary mechanism for NO-induced melanogenesis [9].

α-MSH is reported to regulate melanogenesis via the cyclic adenosine 3′,5′-monophosphate (cAMP) pathway. Downstream melanocortin-1 receptor (MC1R) signaling events mediated by agonist treatment with the α-MSH hormonal peptide include cAMP accumulation [17], activation of the pigmentary transcription factor microphthalmia-associated transcription factor (MITF) [18], increased levels of the major pigmentation protein tyrosinase (TYR), dopachrome tautomerase (DCT) and tyrosinase related protein-1 (TYRP-1) [19]; as well as an increase in dendricity [20], pigment production [19], [21], [22], and cell growth [19], [22], [23].

It is not known whether an NO-induced pathway can affect an α-MSH induced pathway. Recently, Keiichi Hiramoto used UVB to stimulate the eyes and ears of mice, and the results show that the NO-dependent signaling of cellular response to activate the hypothalamopituitary proopiomelanocortin system, thereby enhancing the hypophysial secretion of α-MSH to stimulate α-MSH-receptor responsive cells [24]. But it is not known whether an NO-induced pathway can enhance the ability of melanocytes to respond to α-MSH on melanogenesis in vitro. This is important for us to know in order to enhance the coat color of alpaca.

In this study, we used cultured alpaca skin melanocyte under UV radiation conditions to investigate the effect of the presence or absence of NO (using NG-nitro-l-arginine methylester hydrochloride, L-NAME, a specific NOS inhibitor) on proopiomelanocortin (POMC, α-MSH precursor), MC1R, and melanogenesis-related genes. This can help us to understand the molecular and signaling mechanisms.

Section snippets

Melanocyte culture

All alpaca melanocyte cell cultures used in this study were established in the laboratories of alpaca biology, College of Animal Science and Technology, Shanxi Agricultural University, China [25]. Melanocytes in the culture were maintained in a Melanocyte Basal medium supplemented with 0.2 μg/ml cholera toxin (Sigma), 0.05 mg/ml gentamicin, 2.5 μg/ml fungizone, 50 μg/ml bovine pituitary extract (BPE), 0.5 μg/ml hydrocortisone, 1 ng/ml bFGF, 5 μg/ml insulin, and 10 ng/ml TPA. They are primary cells, so

Nitric oxide assay

About 200 μl of supernatant culture medium was obtained (n = 5 in each group) to assay nitric oxide concentration. The concentration of nitric oxide was 8.819 ± 1.541 μM in control, 25.124 ± 1.233 μM in the UV group, and 6.683 ± 2.864 μM in the UV + L-NAME group. The results show that the UV group can produce more NO than the other groups (p < 0.05). L-NAME can significantly reduce the concentration of NO in alpaca skin melanocytes under UV radiation.

Determination of the key protein associated with pigmentation

We investigated the potential for molecular responses

Discussion

Melanin is synthesized by specific tyrosinase and tyrosinase-related enzymes, which are expressed in melanocytes. The results of our research demonstrate that the TYR and DCT were substantially elevated or decreased following the increase or decrease of the concentration of NO. Sasaki and Roméro-Graillet also got similar results [9], [26]. We suggest the TYR and DCT expression of alpaca skin melanocyte cells relies on NO, which is cultured in vitro under UV condition (Fig. 1, Fig. 2). These

Acknowledgment

This research was sponsored by the China National Natural Science Foundation (Grant Nos. 30571070 and 30671512).

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