The flavonoids apigenin and luteolin suppress ultraviolet A-induced matrix metalloproteinase-1 expression via MAPKs and AP-1-dependent signaling in HaCaT cells

Abstract

Background

Ultraviolet (UV) irradiation causes major changes in skin connective tissues as a result of the degradation of collagen, a major structural component of the extracellular matrix. This process is likely mediated by matrix metalloproteinases (MMPs). Such changes in collagenous skin tissues have been suggested to be causes of cutaneous aging and skin cancer.

Objective

We investigated the protective effects of apigenin and luteolin on immortalized human keratinocytes (HaCaT) against UVA damage. We then explored the inhibitory effects of apigenin and luteolin on UVA-induced MMP-1 and investigated the molecular mechanism underlying those effects.

Methods

HaCaT cells were treated with apigenin and luteolin for the indicated times followed by irradiation with UVA. Those effects were assessed by semi-quantitative PCR, Western blotting and enzymic activity assays.

Results

These two compounds, at concentrations of 1–5 μM, increased the viability of, and inhibited ROS production in HaCaT cells exposed to UVA irradiation. Pre-treatment of HaCaT cells with apigenin and luteolin also inhibited UVA-induced production of the collagenases MMP-1. They also suppressed UVA-induced expression of c-Jun and c-Fos and the phosphorylation of three MAP kinases, upstream modulators of AP-1. Furthermore, the same two flavonoids decreased the UVA-induced influx of Ca²⁺ into HaCaT cells and the phosphorylation of Ca²⁺/calmodulin-dependent kinases (CaMKs).

Conclusion

The results indicate that apigenin and luteolin inhibited UVA-induced collagenolytic MMP-1 production by interfering with Ca²⁺-dependent MAPKs and AP-1 signaling. They may thus be potentially useful in the prevention and treatment of skin photoaging.

Introduction

Skin aging can be attributed to extrinsic and intrinsic (chronological) processes that are commonly manifested by increased wrinkling, sagging, and laxity [1], [2]. Ultraviolet (UV) irradiation causes distinct changes in skin collagenous tissues as a result of the breakdown of collagen, a major component of the extracellular matrix [3], [4], [5]. Exposure of skin to UVA, and occasionally UVB, induces the intracellular generation of large quantities of reactive oxygen species (ROS). ROS-induced molecular damage produces a number of harmful effects on cellular function and homeostasis, while degradation of extracellular matrix (ECM) proteins, such as collagen, by ROS can cause major changes in skin connective tissue [4], [5]. These alterations in the ECM, most likely mediated by matrix metalloproteinases (MMPs), are known to be a cause of the skin wrinkling that characterizes premature skin aging and aged skin [6]. Recently, it was suggested that excessive matrix degradation by UV-induced MMP-1 secreted by various cells (e.g., keratinocytes and fibroblasts cells) contributes substantially to the connective tissue damage that occurs during photoaging [7], [8]. Previously it has been demonstrated that organ culture fluid from either UV-exposed skin or basal cell carcinomas contains large amounts of active MMP-1 as well as smaller amounts of two other collagenases (MMP-8 and MMP-13) [9]. This evidence suggests that the expression of MMP-1 plays a major role in the process of photoaging in keratinocytes.

UV irradiation activates growth factor receptors, which subsequently activate protein kinase signaling pathways, such as the MAPK cascade [10], [11]. MAPK signaling plays important regulatory roles in various cellular processes, including MMP secretion [12]. MAPK activation results in the induction of the transcription factor “activator protein” (AP)-1, which itself regulates the transcription of MMP genes [11]. Oxidative stress contributes to this process by increasing ROS production. ROS influence MAP kinase signaling and thereby contribute to the AP-1-induced up-regulation of MMP-1 [13].

It has recently been suggested that Ca²⁺ regulates the expression and/or activation of MMPs. Increased extracellular Ca²⁺ levels induce MMP-9 gene expression in human keratinocytes [14], [15], while inhibiting the influx of Ca²⁺ decreases the levels of MMP-1 mRNA [16]. Modulation of intracellular Ca²⁺ levels can affect the secretion of MMP-1 from migrating keratinocytes [17]. Recent data suggest that the influx of Ca²⁺ through “transient receptor potential vanilloid-type 1” (TRPV1) channels is key in heat shock-induced MMP-1 expression in HaCaT cells [18], while Lee et al. reported that it is essential to UV-induced MMP-1 expression in HaCaT cells [19]. Ca²⁺/calmodulin (CaM) is a Ca²⁺-binding protein that has been implicated in various cellular functions, including cell growth/proliferation and migration [20]. Ca²⁺/CaM does not itself display catalytic activity, but regulates the activity of a number of Ca²⁺/CaM-dependent enzymes, including Ca²⁺/calmodulin-dependent kinase (CaMK) [21].

Polyphenolic flavonoids, found in many fruits and vegetables, are natural antioxidants that scavenge various types of radicals in aqueous and organic environments [22], [23]. Several studies have revealed that polyphenols show promise in reducing the risk of skin diseases, apparently as a result of their antioxidant properties [24], [25], [26], [27]. However, how flavonoids protect cells and tissues from UV-induced oxidative damage remains unclear. Intense interest has focused on the beneficial effects of dietary polyphenols, and specifically their anti-oxidative and anti-inflammatory activities, on the skin [28], [29].

Apigenin and luteolin are two common dietary flavonoids that are found in a large number of fruits and vegetables, including parsley, onions, wheat sprouts, chamomile, seasonings, tea, and orange [30], [31], [32]. They have gained much attention due to their health benefits and their ability to inhibit the development of different cancers (they are potent inhibitors of the proliferation of skin, prostate, colon, breast, and thyroid cancer cells) [33], [34], [35], [36]. They also possess anti-inflammatory and antioxidant properties [37], [38], [39]. Moreover, a recent study highlighted their ability to inhibit MMPs activity and COX-2 expression in UV-irradiated dermal fibroblast and keratinocytes [35], [40]. Although apigenin and luteolin have been tested in various bioactivity assays, the molecular mechanisms by which they modulate the expression of MMPs during the photoaging of human keratinocytes have not been investigated. In the present study, we assessed the effects of apigenin and luteolin on UVA-induced collagenase activation, and attempted to identify the upstream signaling pathways involved.