Heat shock proteins (HSP): dermatological implications and perspectives

ARTICLE

ejd.2011.1530

Auteur(s) : Wagner Vidal Magalhães¹ wmagalhaes@usp.br, Marcelo Fábio Gouveia Nogueira², Telma Mary Kaneko¹

¹ Laboratório de Controle Biológico de Qualidade de Medicamentos e Cosméticos - Universidade de São Paulo, Av. Prof. Lineu Prestes 580, Bloco 13, São Paulo, Brasil

² Laboratório de Micromanipulação Embrionária - Universidade Estadual Paulista Júlio de Mesquita Filho, Avenida Dom Antônio, 2100, Assis, Brasil

Reprints: W. Vidal Magalhães

Heat shock proteins (HSPs) are the most abundant proteins in the biosphere [1]. The fact that they show remarkable evolutionary conservation suggests they are essential for basic cellular function. A number of studies have shown that the expression of these proteins has a close relationship with life history, exerting influence on biological phenomena such as stress resistance [2, 3] and longevity [4-6]. HSPs are identified and classified into families according to their molecular masses measured in kilodaltons (kDa). In table 1, the major HSPs and their functions are represented.

Table 1 Major HSPs and their functions.

The initial step for the discovery of this broad group of shock proteins occurred in 1962, when Ritossa [7] incidentally observed a new pattern of chromosomes thickening in Drosophila salivary gland cells exposed to high temperatures. Besides heat shock there are different factors responsible for the expression of HSPs. The most studied category of stimuli corresponds to environmental stress, which are the best known inducing agents, such as radiation [8-10], oxidative damage [11, 12], heavy metals [13, 14] and even heat stress [15, 16]. Many of these stressors share the property of being proteotoxic, i.e. elements that adversely affect the correct conformation and, consequently, functioning of proteins [17]. In such circumstances, HSPs are synthesized largely as a response which aims at minimizing cell damage, ensuring the survival of the cell or organism, and also inducing subsequent stress resistance. This inducible cellular tolerance was firstly reported after heat shock and was thus called thermotolerance [18].

There are also constitutively expressed HSPs that under normal conditions act as molecular chaperones. Chaperones exert activity in the synthesis, folding and transport of newly synthesized proteins (see the review in [19]), reducing the likelihood of inappropriate protein interactions, which can result in pathological changes in a given organism (see the review in [20]).

HSPs in skin

In recent years, numerous studies have demonstrated the protective effects of HSPs on different organs subjected to different stressful conditions. In skin, cells of both dermis and epidermis express HSPs [21, 22], which confer resistance to damage caused by stressors such as sunlight exposure [23].

However, these proteins show expression patterns that vary with the stressor and cell type. Keratinocytes, for example, constitutively express HSP 72, which is consistent with the need for these cells to protect themselves against numerous forms of environmental stress to which they are subjected daily and constantly [24]. In fibroblasts, on the other hand, HSP 72 expression is almost undetectable in normal conditions [25]. Other HSP families - such as HSPs 27, 60, 90 and 110 - are also normally present on skin, predominantly in the epidermis [26].

Each HSP has essential and specific functions in the skin - the HSP 27 stabilizes the actin cytoskeleton [27] and is involved in keratinocyte differentiation [28], whereas HSP 47 is associated with collagen synthesis in fibroblasts [29]. In recent decades, many studies have investigated the expression of different HSP families in various situations, such as aging and tissue repair, in order to unravel the role of these proteins during such events.

HSPs in photoaging

Photoaging is mainly the result of UV-induced damage of the dermal connective tissue of the skin [30]. DNA damage and oxidative stress are the initial molecular events that lead to the majority of clinical and histopathological features of photoaging, as a result of UV radiation exposure [31].

UV-B radiation induces the synthesis of photoproducts, dimers, between adjacent pyrimidine bases in DNA strands [32]. If not repaired, the dimers are propagated over cell multiplication and may result in tumor formation [33]. These lesions in DNA also contribute to the process of skin aging by signaling the release of metalloproteinase-1 (MMP-1) from fibroblasts, the enzyme responsible for collagen degradation [34].

Moreover, the skin is one of the organs most affected by oxidative stress, by exposure to external agents such as air pollution [35] and radiation [36], as well as by normal metabolism of cells [37, 38]. In all cases, the production of reactive oxygen species (ROS) occurs, which are capable of damaging biological sites, such as lipids, proteins and DNA. Damage to any of these components can interfere seriously with the normal functions of cells and can result in pathological processes such as inflammation and cancer [39].

The development of characteristic changes in collagen and elastic fibers, observed in photoaging, also has a direct relationship with the generation of ROS. According to a study by CHOI et al. (2009) [40], the increase of tropoelastin (precursor molecule of elastin) mRNA levels, due to the generation of ROS, contributes to the accumulation of abnormal elastic material. This phenomenon, known as solar elastosis, is considered the most evident pathological change in photodamaged skin. Moreover, oxidative stress induces the synthesis and activation of metalloproteinases [41, 42] and inactivates inhibitors of these enzymes [43].

In this context, it is important to highlight HSP 72 as an agent of great potential in the fight against photoaging. This protein was able to reduce the accumulation of oxidized and glycoxidized proteins of human fibroblasts in in vitro tests [44]. Such capacity may be due, at least in part, to its ability to regulate cellular redox status by modulating glutathione peroxidase and glutathione reductase activities [45].

Furthermore, heat shock (a potent HSP inducing stimulus) has been described to attenuate UVB-induced cell death in human skin cells [46]. The HSPs role during this protection process was observed in one study that demonstrated that inhibition of HSP 72 expression nullifies the protective effect of a prior hyperthermia on subsequent UV-induced damage on human A431 keratinocytes [47]. Jantschitsch et al. (2002) [48] observed that the heat preconditioning of human melanocytes, keratinocytes and fibroblasts was also able to slightly increase the rate of repair of UV-induced thymine dimers 3 hours after exposure to UV-B, compared to controls.

Besides HSP 72, another stress protein plays a fundamental role in adaptive responses against oxidative stress. HSP 32, also known as heme oxygenase, is considered an antioxidant enzyme because of its protective role against the toxic effects of free radicals generation. This enzyme catalyzes the degradation of free heme (a potent oxidizing agent) in Fe³⁺, carbon monoxide and biliverdin, which is converted under the action of biliverdin reductase to bilirubin [49], a pigment that has antioxidant properties [50].

The role of HSPs during the aging process is also highlighted by the fact that induction of these molecular chaperones is intimately related to increased longevity. Tatar and colleagues (1997) [51] found that heat-induced expression of HSP 70 increased Drosophila melanogaster lifespan at normal temperatures. Also, it is known that HSP 90 is essential for the proper functioning of telomerase, the enzyme that regulates cellular lifespan [52].

Despite the enormous importance of heat shock proteins in the aging process, in various experimental models the stimulation potential of HSP synthesis in senescent cells submitted to a stressor or inducer was shown to be lower than in young cells. Nizard et al. (2004) [53] demonstrated that the induction of HSP 47 expression in response to application of Salix alba extract in cultured fibroblasts is significantly more severe in young cells. In addition, a decreased level of induction of HSP 72 in aged skin cells subjected to heat stress (45̊C for 1 hour) was observed [54].

HSPs in skin cancer

Currently, skin cancer is considered the greatest incident cancer type worldwide, predominantly in Caucasians. The total number of procedures for skin cancer increased 76.9% in the Medicare fee-for-service population during the period between 1992 and 2006 [55]. A 2004 survey showed the existence of 2.9 million new cases and an average of 1.7 million deaths annually, considering the population of 25 European countries [56].

Skin cancer is characterized by abnormal and uncontrolled growth of cells that make up the skin. UV is experimentally and epidemiologically proved as the main “causal agent” of skin cancer [57]. Notably, the prevalence of these lesions is directly related to the individual's age [58, 59] and it may be conjectured that this fact is due to cumulative exposure to radiation. The protection of epidermal cells against damage by UV radiation is mediated by numerous mechanisms such as DNA repair, activation of antioxidant enzymes, increased melanin and the expression of some HSPs. It is known that during UV exposure the transcription factor HSF-1 (responsible for controlling the expression of HSP 72) is activated, resulting in increased expression of HSP 72 in keratinocytes [60].

A group of researchers demonstrated the ability of HSPs to protect against skin cancer [61]. In this study, it was reported that overexpression of HSP 27 in transfected cell line A431 (human epidermal carcinoma) injected into nude mice was able to slow tumor development. Also, clinical trials in melanoma have demonstrated that HSP-peptide complexes (HSPPC-96) derived from autologous tumors can be used as a vaccine to treat melanoma [62].

In contrast to HSPs’ therapeutic potential in cancer treatment, it has also been shown that HSP 72 expression favors the survival of cancer cells [63]. In this study, the authors observed that after neutralization of HSP 72 by a peptide, mouse melanoma cells (B16F10) became more susceptible to apoptosis.

HSPs in wound healing

The wound healing process comprises several complex interdependent phases. These phases consist of three important steps: inflammation, granulation tissue formation with extracellular matrix deposition (proliferation) and remodeling (maturation) [64]. Failure or prolongation of these phases can result in delayed healing or non-closure of surgical wounds, which represent a significant cause of morbidity and mortality that implies an increased cost to health systems [65, 66].

In this context, it is important to emphasize the role of HSPs. During tissue repair, several cells inside or near the lesion are subjected to a wide variety of stressful conditions such as dehydration, exposure to oxygen, interruption of blood supply and decreased glucose levels [67]. Laplante et al. (1998) [68] examined the expression of HSPs 27, 60, 72 and 90 during the repair process, and observed changes in the expression pattern of these proteins in such situations. In the thickened epidermis, HSP 70 showed a reduced expression, HSP 60 was induced in low suprabasal and basal cells, whereas HSP 90 and HSP 27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells.

HSP 47, HSP 72 and HSP 32 are among the most important HSPs during tissue repair. HSP 47, located in the endoplasmic reticulum lumen, participates in the early stages of synthesis, folding and assembly of the trimeric structure of procollagen, a precursor molecule of collagen [29, 69]. In addition, it has been shown that overexpression of HSP 47 is responsible for the overproduction of collagen, the predominant phenomenon in the formation of keloids [70].

HSP 72, also known as HSP 70, is a cytosolic protein that acts in different forms during wound healing. Besides acting as a molecular chaperone and helping with homeostasis and cell survival, the expression of HSP 72 is related to the increased phagocytic ability of macrophages during healing [71]. Constitutively, this protein is little expressed in the epidermis whereas, in the dermis, its expression is not observed before or after an injury. However, its presence is remarkable in epidermal cells near the injured area [72]. One study has also shown that delivery of HSP 72 into renal tubular cells confers anti-inflammatory action due to its ability to block activation of nuclear factor NF-kB [73], responsible for activation of several pro-inflammatory molecules.

After a tissue injury, increased expression of HSP 32 occurs in the early days, being probably important for cells to support oxidative stress due to resulting inflammatory response [72]. Increased expression and activity of HSP 32, depending on the treatment of C57BL6 mice with tin chloride, resulted in a significant acceleration of wound healing and attenuation of the inflammatory response [74].

Interestingly, application of recombinant HSP 90 in a carboxymethylcellulose cream enhanced skin wound healing during days 5–13, with an overall 30% improvement of mice wound healing time [75].

Pharmacological inducers

Given the diversity of cytoprotective properties of HSPs, there is enormous interest in discovering and developing pharmacological agents capable of safely modulating the expression of these proteins. Despite the use of pharmacological inducers which flee the field of emergencies, due to the fact that cells require a period of time to fully develop the production of HSPs, such agents find applications in elective surgery [76] or in the prevention of skin disorders through their incorporation into cosmetic products.

As already mentioned, there are numerous agents capable of elevating the expression of HSPs. However, the vast majority of these inducers, such as heat shock, UV radiation or ethanol exhibit significant cytotoxicity and thereby compromise cell viability, which explains the induction of these agents. However, the induction of HSPs by known drugs such as geranylgeranylacetone [77] and bimoclomol [78] has been verified, which demonstrably do not constitute a form of stress to cells. The ability of plant extracts and compounds to induce HSPs has also been verified, such as the extract of Salix alba, which can raise levels of HSP 47 in fibroblasts [53], and pigment curcumin, derived from Curcuma longa, which induced expression of HSP 32 in a non-toxic manner in normal adult skin fibroblasts [79]. In addition, paeoniflorin, one of the major constituents of a herbal medicine derived from Paeonia lactiflora, showed activity in inducing HSP 27, 40 and 72 in in vitro tests [80].

Conclusion and perspectives

Recent studies have demonstrated the therapeutic potential of manipulating HSP expression, including, most importantly, vaccines against tumors and cell protection against lethal damage caused by stress agents.

With regard to dermatology, protection against environmental stresses such as UV radiation, heat and pollution, prevention and repair of the effects of skin aging, and the optimization of the wound healing process are possible application fields for topical and systemic HSP-inducer compounds. Induction of HSP 72, for example, find applications in anti-aging products, due to its ability to reduce the accumulation of oxidized and glycoxidized proteins in skin cells, and also to decrease cell damage induced by UVB. In turn, HSP 32 is a key enzyme that exerts an antioxidant role, protecting cells against oxidative damage, presenting potential for use in products that reduce oxidative damage caused by sun exposure. HSP 47 is another protein of huge dermatological importance because it is related to collagen synthesis by fibroblasts, and is thus a target protein both for cosmetics and for skin wound healing products.

Besides the use of expression inducers, encapsulation vehicles, such as liposomes, emerge as a likely alternative for protection of skin cells by HSPs for topical application in order to generate direct cytoprotective effects.

Considering the search for eternal beauty built into the current cultures in different nations and social classes, as well as the increased demand for multi-functional cosmetic products, the direct availability or induction of HSPs by products of this genre comes with excellent success prospects, both for the cosmetics market and for dermatological medicine. As an example, Crème Reminéralisante^® marketed by Anna Pegova can be mentioned. According to the product description, its anti-aging property is due to its ability to stimulate the synthesis of heat shock proteins [81]. Another example, Langerhans cells treated with RonaCare Ectoin^® (Merk^®) showed, under stress conditions, faster formation of HSP 72 in comparison to untreated cells [82].

Because of HSPs’ remarkable protective and preventive capacity, the development and evaluation of active compounds capable of inducing their expression should be considered as essential goals of the current scientific community.

Disclosure

Financial support: none. Conflicts of interest: none.

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