Exogenous nitric oxide enhances the synthesis of type I collagen and heat shock protein 47 by normal human dermal fibroblasts

https://doi.org/10.1016/j.jdermsci.2005.08.004Get rights and content

Summary

Background

It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified.

Objective

In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen αI S(1) mRNA/protein and heat shock protein 47 (HSP47).

Methods

The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR.

Results

NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen αI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner.

Conclusions

These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen αI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.

Introduction

Nitric oxide (NO) has been identified as an endothelium-derived relaxing factor [1] and has been shown to possess various physiological and pathophysiological activities. It is well known that in cells of several tissues NO is produced by the catalytic action of NOS using l-arginine as a substrate. NOSs have been identified in three different isoforms. Two of those isoforms are constitutive NOSs located in neuronal cells (ncNOS) or in the endothelium (ecNOS) [2]. The third isoform is inducible (iNOS), which is typically induced by inflammatory and immune stimuli such as inteferon-γ, tumor necrosis factor-α and interleukin-1β [3].

NO is produced by almost all types of skin cells, keratinocytes, melanocytes, Langerhans cells, and fibroblasts [4]. In the epidermis, NO causes several biological effects including allergic reaction [5], immunomodulation [6], the formation of sunburn erythema and edema [7], and melanogenesis [8]. Some reports have been published on the contribution of NO to wound healing [9], [10], [11]. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in NO at wound site. Treatment with l-arginine improved healing in diabetic animals accompanying with increases of nitrite/nitrate level in wound fluid. In addition, the elevation of mRNA of procollagens I and III was reported [10]. Also, treatment with NO donor to wound area was reversed impaired healing in diabetics with increasing collagen synthesis [11]. In wound healing, it has been established that NO is an important factor. Witte et al. also reported that exogenous NO enhanced type I collagen synthesis by normal fibroblasts at the post-translational level in vitro study [12]. However, the precise feature of NO-induced collagen synthesis has not been identified.

It was reported that HSP47 plays a critical role in the process of collagen synthesis, because it works as a collagen-specific molecular chaperone [13]. Therefore, the possibility was considered that NO stimulates HSP47 expression concomitantly with collagen synthesis.

Thus, the purpose of this study was to establish the mechanisms of exogenous NO on collagen synthesis by normal human dermal fibroblasts, including effects on HSP47 expression. Our results indicate that NO generated by NO donors increases collagen synthesis and HSP47 protein levels. This is the first report that exogenous NO increases HSP47.

Section snippets

Reagents and cell culture

Bovine serum albumin (BSA) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), methylene blue (MB), N2,2′-O-dibutyryl-guanosine 3′,5′-cyclic monophosphate (dibutyrylcGMP) and anti-mouse IgG antibody were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Dulbecco's modified Eagle medium (DMEM) was obtained from Nikken Bio

Effects of exogenous NO on collagen synthesis by dermal fibroblasts

To address the effects of NO on type I collagen synthesis, we examined whether SNP and SNAP, which are donors of NO, stimulated collagen synthesis by human dermal fibroblasts. Both types of NO donors significantly accelerated the production of type I collagen by cultured dermal fibroblasts in a dose-dependent manner (Fig. 1). To elucidate whether the stimulation by a NO donor, SNP, on collagen production was due to NO, the stimulating effect of SNP was examined in the presence of methylene blue

Discussion

Witte et al. reported that exogenous NO did not affect collagen type I or III gene transcription, but increased collagen synthesis [11]. However, our results show clearly that exogenous NO, generated by SNP or SNAP, significantly accelerates the synthesis of type I collagen by human dermal fibroblasts at both the transcriptional and translational levels. Furthermore, the action of NO on stimulating collagen synthesis could be abolished by the addition of MB, an NO inhibitor, while dibutyryl

References (19)

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