Signal transduction pathways in human epidermis

ARTICLE

Auteur(s) :, Lars Iversen^*, Claus Johansen, Knud Kragballe

Dept of Dermatology, University Hospital of Aarhus, PP Orumsgade 11, 8000 Aarhus C, Denmark

accepté le 21 Septembre 2004

Membrane receptors

General features of cytokines are their pleiotropism and redundancy. Pleiotropism refers to the ability of one cytokine to act on different cell types which reflects that different cell types may have the same type of receptors on their membrane surface. Redundancy refers to the property of multiple cytokines having the same functional effect which can be explained by the fact that the same receptor or receptor type can bind different cytokines.

Different ways of classifying cytokine membrane receptors have been applied. Membrane receptors can be separated depending on the dominant signal transduction pathway that is activated or into groups expressing or lacking intrinsic enzymatic activity. Here the membrane receptors will be separated into six general classes (table 1( Table 1 )).

The IL-1R bears only little homology with other cytokine receptors, and therefore it constitutes its own receptor class. It binds both IL-1α and β, is expressed on almost all cell types and is the major receptor for IL-1 mediated responses. The dominant signal transduction pathway is activation of NF-κB and in part AP-1 (see below). The IL-1R has no intrinsic enzymatic activity but upon activation the intrinsic part of the receptor associates with a cytoplasmic sereine-threonine kinase called IL-1 receptor-associated kinase (IRAK) which initiate the signaling responses.

TNFα is also an important mediator of cutaneous inflammation and has been shown to be synthesized in a number of different cell types including human keratinocytes, lymphocytes, fibroblasts and monocytes [1]. There are two distinct TNF receptors, TNF-RI with a molecular mass of 55 kD and TNF-RII with a molecular mass of 75 kD. The soluble form of TNFα is homotrimers and trimerization of the TNF receptor family members is also required for induction of signal transduction [2]. The TNF receptors do not exhibit any intrinsic enzymatic activity but cytokine binding members lead to the recruitment of proteins called TNF receptor associated factors (TRAFs) to the cytoplasmic domain of the receptor and subsequently activation of transcription factors, especially NF-κB and AP-1. The strong anti-psoriatic effect of TNFα inhibitors underscores the importance of TNFα mediated signaling.

The hematopoietin receptor superfamily is the largest of the cytokine receptor families. It is a group of structurally related type I membrane-bound glycoproteins characterized by one or more copies of a domain with two conserved pairs of cystein residues. A number of cytokines including IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, GM-CSF, G-CSF and growth hormone signal through the hematopoietin receptor family [3]. The class I receptors have no enzymatic activity themselves but after ligand binding the cellular recptors couple to cytoplasmic protein tyrosine kinases which induces enzymatic activity. The main signal transduction pathway for this class of cytokine receptors is the Jak/STAT pathway, although the class I receptors are less specific than the class II receptors.

The class II cytokine receptors are a second major class of cytokine receptors. Today there are 12 known members of this class II cytokine receptor family: four receptor subunits (IFN-αR1, IFN-αR2, INF-γR1 and IFN-γR2) for the type I (INF-α and INF-β) and type II (INF-γ) INFs, two IL-10 receptor subunits (IL-10R1 and IL-10R2), four receptors utilized by IL-10 related cytokines (IL-20R1, IL-20R2, IL-22R1 and IL-22BP), the tissue factor (TF) that binds FVIIa and one currently orphan receptor [4]. Class II receptors are composed of two distinct receptor chains denoted R1 and R2. Ligand binding to the R1 type subunit induces oligomerization of the receptor subunits, recruitment of particularly STAT transcription factors followed by Jak or Tyk mediated tyrosine-phosphorylation and activation of the STAT transcription factor. The role of the R2 type subunit is to initiate the signal transduction events [4].

More than 30 members of the TGF-β family have been identified. The TGF-β family members are made as precursors that are biologically inactive. Once activated by cleavage of a large pro-domain, TGF-β binds to the ligand binding type II receptor which then associates with a type I receptor. The type I/type II receptor complex possesses serine/threonine kinase activity resulting in down stream signal transduction primarily through a family of cytoplasmic Smad transcription factors that translocate to the nucleus and regulate gene transcription [5].

Chemokines represent a large family of structurally related small (8-14 kDa) polypeptide signaling molecules [6]. They bind to the chemokine receptor family which are seven transmembrane G protein-coupled receptors [7]. The long list of chemokines and chemokine receptors has been extensively reviewed by Zlotnik et al. [6]. The chemokines and their respective receptors are grouped into four subfamilies, the CXC, CC, C and CX₃C. There is a considerable redundancy and promiscuity in chemokine signaling because many chemokines share the same receptors. After ligand binding the chemokine receptor associates with intracellular heterotrimeric G proteins [6]. Chemokine receptor induced G-protein linked signal transduction results in downstream activation of a number of signaling pathways such as e.g. the PKC and the MAPK signaling pathways.
Table 1 Cytokine receptors

JAK/STAT pathway

In the nucleus activated STAT acts as a transcription factor by inducing gene transcription through binding to specific DNA response elements (( figure 1 )). STAT dependent target genes influence a number of different cellular functions including cellular growth, survival, apoptosis, host defence and regulation of stress and differentiation as demonstrated in knockout studies of STAT family proteins in mice [8]. In normal cells, ligand dependent activation of the STATs is a transient process lasting for minutes to hours. In contrast inappropriate persistent STAT activation has been associated with oncogenesis [9].

The expression of the JAK and STAT family proteins has been determined in human epidermis by immunolocalization [10]. Especially the role of STAT3 has been investigated in the epidermis. Epidermal cells and keratinocytes that are defective in STAT3 gene expression show impaired growth-factor dependent in vitro migration [11] and it was also demonstrated that while the development of the epidermis and hair follicles appeared normal, hair cycle and wound healing were severely compromised in STAT3 mutant mice. These results indicate that STAT3 is essential for skin remodeling such as wound healing and hair cycle regulation.

Abnormal signaling in the JAK-STAT signaling pathway has also been suggested in psoriasis. STAT1 activation in response to IFN-γ in keratinocytes from psoriasis has been shown to be reduced compared to normal kerationocytes [12], and an aberrant response to IFN-γ may be important for the altered apoptosis and desquamation seen in psoriasis.

Non-receptor serine/threonine kinases

Protein kinase C (PKC)

The various PKC isozymes can be activated by a number of different stimuli such as e.g. UV radiation and TPA and repeated PKC activation in mouse skin keratinocytes with either one of these two stimuli has led to the development of skin carcinogenesis. Therefore a number of studies trying to determine the role of the various PKC isoforms in the development of skin cancers have been carried out. Taken together these studies indicate that individual PKC isoforms have opposite effects on skin carcinogenesis, but further studies are needed to explain and understand their precise role [15].

MAPKs

At least four different and distinctly regulated groups of MAP kinases have been described. These include the extracellular signal-regulated protein kinases (ERK) [16], the c-Jun NH₂-terminal kinases (JNK) [17], the p38 MAP kinases [18] and ERK5 [19] (( figure 1 )). The MAPK cascade represents a family of protein kinases that utilizes sequentiel phosphorylation of specific kinases to regulate the various cellular processes. In the keratinocytes MAPKs play a key role in regulating cell differentiation, apoptosis and inflammation [20].

ERK1+2

Because ERKs like other MAPKs are activated by phosphorylation, the protein phosphatases dephosphorylating MAPKs are key elements in controlling ERK activity.

Once ERK1 and 2 have been activated they can target both cytoplasmic proteins, membrane proteins, cytoskeletal proteins and nuclear proteins. An important role of ERK activation is regulation or modulation of gene expression which is mediated in two ways. ERK1 and 2 can target a group of protein kinases including RSK 1-4, MAPKAP kinase 2 and 3 and Mnk1 and 2 which enhance gene expression through increasing the accessibility of DNA in the cell [21]. ERK 1 and 2 also target the mitogen- and stress-activated protein kinases (MSK1 and 2) which is a family of serine/threonine protein kinases that regulate the activity of a number of different transcription factors. However, ERK1 and ERK2 also phosphorylate transcription factors directly and thereby stimulate their activity.

ERK1 and ERK2 have both been demonstrated in the epidermis as well as in normal human keratinocytes in vitro and have been suggested to play a key role in regulating keratinocyte proliferation, differentiation and survival [20, 22].

Increased ERK1 and ERK2 expression in the basal and lower suprabasal layer of lesional psoriatic skin has been demonstrated [23] as well as an increase in the activated form of ERK [24] and in a very recent study ERK activation was demonstrated to play an important role in epidermal hyperproliferation and skin inflammation [25].

ERK activation seems also to be important in a number of human cancers. Numerous solid tumors are known to express constitutive levels of activated ERK1 and ERK2 and ERK activation is critical for a large number of Ras-induced cellular responses. Interestingly the Ras family of proteins is activated by mutation in approximately 30% of human cancers [26].

JNK1-3

TNFα can activate the JNK signaling pathway through binding to TNF receptors and, conversely, the expression of the TNF gene is regulated by JNK activity. Glucocorticoids have been shown to specifically repress the activity of the JNK signaling pathway. Dexamethasone inhibits TNFα and lipopolysaccharide (LPS) induced activation of JNK whereas no regulation was seen of the ERK and p38 MAPK pathway [29].

JNKs exert their effects only on transcription factors in the nucleus. This is in contrast to the other MAPKs which phosphorylate targets both inside and outside the nucleus. Activation of the JNK signaling pathway plays an important role in regulating apoptosis as well as tumorigenesis and inflammation.

In the skin JNK1 and JNK3 have been demonstrated in cultured normal human keratinocytes in vitro, and immunohistochemical analyses of normal human skin have revealed that phosphorylated active JNK is expressed in the nuclei in the suprabasal-granular cell layer [30] indicating that JNK participate in regulation of gene transcription in differentiating normal human keratinocytes.

UV light often affects the human skin. It acts as an initiator of cellular stress and activates signal transduction resulting in regulation of transcription factors. In keratinocytes only UVB and UVC activate JNK while in other cell types UVA, UVB and UVC do [31]. These findings demonstrate that in the epidermis UV light acts as a specific agent regulating highly specialized responses which may be of importance for the role of JNK in regulating tumor development. Studies with JNK1-deficient mice have suggested that JNK1 is a crucial suppressor of skin tumor development [32].

p38 MAPK

p38 MAPKs are predominantly activated by inflammatory cytokines like TNFα and IL-1 and they play a key role in regulating the cellular responses to these cytokines. However, p38 MAPKs can also be activated by a number of physical and chemical stresses like oxidative stress and UV irradiation [39].

p38δ has been shown to play an important role in inducing keratinocyte differentiation [76] and in a very recent study data strongly suggested p38δ as the major p38 isoform in driving the expression of the keratinocyte differentiation marker involucrine [40]. p38δ has also been ascribed a key role in the induction of apoptosis in keratinocytes [38].

A number of downstream targets of p38 have been demonstrated. In the nucleus, p38 regulates the activity of a number of transcription factors including ATF-1/2, Elk-1, p53, NF-κB and AP-1 [21]. An inflammatory stimulus has been shown to induce p38α and/or p38β dependent phosphorylation of histone H3 in the promotor region of a subset of NF-κB dependent cytokine and chemokine genes resulting in increased NF-κB recruitment and subsequently increased gene transcription [41]. This demonstrates that p38 plays an important role in regulating inflammatory and immune responses. Other substrates downstream of p38 include different protein kinases e.g. MAPKAP kinase-2, MAPKAP kinase-3, MAPKAP kinase-5 MSK 1 and 2 and MNK1 [42-45] which act both at the transcriptional and the translational level as well as acting in a feedback mechanism by modulating p38 activity.

The expression and regulation of these downstream targets of p38 MAPK have not been investigated in human skin although it is intriguing to speculate that an imbalance in these protein kinases may be of importance in the pathogenesis of various skin diseases.

ERK5

ERK5 is expressed in many human tissues, but most abundantly in heart and skeletal muscle [19]. It has never been studied in human skin but it has been shown to be expressed in mast cells [46] and in fibroblasts [47].

Similarly to the ERK1/2 pathway ERK5 also activates a distinct set of transcription factors implicated in cell cycle regulation. ERK5 contributes to AP-1 activation by stimulating the expression of genes encoding AP-1 components such as c-jun and c-fos [48]. NF-κB has also been demonstrated as a downstream target of ERK5 and it has been suggested as an integration point for ERK5 and ERK1/2 signaling [49]. Most recently the ERK5 cascade has been shown to induce transcriptional activation of the cyclin D1 gene [50]. Cyclin D1 is expressed in keratinocytes and has been suggested to mediate the proliferation of stem cells in the epidermis into more differentiated transient amplifying cells in the suprabasal layer [51]. Furthermore cyclin D1 overexpression is frequently an early step in neoplastic transformation. Because cyclin D1 is a novel target of the ERK5 cascade it may be interesting to further investigate the role of ERK5 in cancers such as squamous cell carcinomas involving cyclin D1 dysregulation.

Transcription factors

AP-1

AP-1 is not a single protein, but a dimer formed of proteins belonging to the Jun (c-jun, JunB, JunD), Fos (c-fos, FosB, Fra-1 and Fra-2), Maf (c-Maf, MafB, MafA, MafG/F/K and Nrl) and ATF (ATF2, Lrf1/ATF3, B-ATF, JDP1, JDP2) subfamilies [52]. This review will focus mainly on Jun and Fos.

The Jun and Fos proteins are known as early responsive genes which are rapidly but transiently transcribed in response to extracellular stimuli such as growth factors, cytokines, neurotransmitters, bacterial and viral infections, and a variety of physical and chemical stresses [52]. These newly synthesized AP-1 family proteins then dimerize to form the AP-1 transcription factor either as Jun-Fos heterodimers or Jun-Jun homodimers.

AP-1 has been suggested to play a pivotal role in regulating epidermal homeostasis. The expression pattern of Jun and Fos proteins has been studied in both mouse and human epidermis [53, 54]. In human epidermis c-jun was found only in the granular layer whereas c-fos was found both in the spinous and granular layer but not in the basal layer. JunB and JunD were ubiquitously expressed in all epidermal layers. This distinct expression pattern of the AP-1 subunits in the epidermis as well as the expression of a number of target genes including keratin 1 and 5, involucrine and transglutaminase indicate that AP-1 may play a pivotal role in normal and pathological skin physiology although this could not be confirmed in a knock out mouse model. Mice lacking c-fos, Fos or JunD did not show any specific skin phenotype [55].

Recently a decreased AP-1 DNA binding activity has been found in lesional psoriatic skin compared to non-lesional and normal skin (own results, in press Br J Dermatol) indicating a role of AP-1 in the pathogenesis of psoriasis. This is further supported by the finding that topical treatment of lesional psoriatic skin with the vitamin D analogue, Calcipotriol leads to a normalization of the AP-1 DNA binding activity preceding the morphological normalization of the skin.

AP-1 also play a role in skin carcinogenesis [52]. Inhibition of malignant transformation of the skin is observed in mice expressing a dominant negative c-jun transgene. However, transgenic overexpression of c-jun alone does not increase skin tumor incidence. Skin carcinogenesis requires additional activation of the AP-1 transcription factor [52].

NF-κB

In resting cells NF-κB is usually retained inactive in the cytoplasm through binding to a member of the NF-κB inhibitor protein family which contain seven members IκBα, IκBβ, IκBγ, IκBε, Bcl-3 and the precursor Rel proteins p100 and p105 [59]. A wide variety of agonists including the pro-inflammatory cytokines TNFα and IL-1 have been shown to activate NF-κB. Once activated the NF-κB dimer translocates to the nucleus where it binds to specific response elements in the promotor region of the various target genes [60].

NF-κB is ubiquitously expressed in almost all tissues investigated. While first discovered as a key regulatory factor of immune and inflammatory responses NF-κB is now recognized as an important player in controlling cellular growth and homeostasis including epidermal proliferation and differentiation [61].

Recently it was shown that NF-κB activation in leukocytes during the onset of inflammation is associated with pro-inflammatory gene expression whereas such activation during the resolution of inflammation was associated with the expression of anti-inflammatory genes and genes important for the induction of apoptosis [62]. Similarly, a change in the predominant NF-κB dimer was seen from 6 to 48 hours after the onset of inflammation.

An inhibition of NF-κB has been suggested as part of the mechanism of action of several anti-inflammatory drugs. Glucocorticoids have immunomodulatory effects and part of these effects have been ascribed an inhibitory effect on NF-κB activation [63, 64]. Cyclosporin A which among other indications is used for systemic treatment of inflammatory skin diseases has also been shown to inhibit NF-κB activation in an animal model with acute hypovolemic shock [65]. Recently, it was demonstrated that dimethylfumarate selectively prevents the nuclear entry of activated NF-κB, and it was suggested that this may be the basis of its beneficial effect in psoriasis [66]. An inhibition of NF-κB activation has also been suggested as the key mechanism for anti-TNFα antibodies effective in psoriasis [67].

In keeping with these observations NF-κB has also been ascribed a key role in the pathogenesis of a number of different skin disorders. Disturbances in NF-κB activation may play a pivotal role in the pathogenesis leading to psoriasis. Results from our group have shown that there is an increase in NF-κB binding to the κB motif in the IL-8 promotor region and a decrease in NF-κB binding to the κB motif in the p53 promotor region in lesional psoriatic skin compared to non-lesional psoriatic skin which is also reflected by an increase in IL-8 expression and decrease in p53 expression. These results demonstrate that NF-κB regulation is very complex, and that there is a high degree of specificity of the genes transactivated by NF-κB in psoriasis. Others have also found disturbances in NF-κB activation in psoriatic keratinocytes [68].

Recently deletion mutations of the NEMO/IKKγ gene were shown to account for most cases of incontinentia pigmenti [69]. The mutation in NEMO/IKKγ results in an inhibition of NF-κB signaling and in male NEMO/IKKγ knock out mice NF-κB activation by proinflammatory cytokines was completely blocked [70].

The involvement of NF-κB in sunburn reactions has also been demonstrated. Because most incident solar UV radiation is absorbed in the epidermis and dermis, the effect on the NF-κB pathway is of particular interest. UV irradiation leads to NF-κB activation and in a very recent publication UVA irradiation of human skin fibroblasts resulted in a prolonged iron-dependent activation of NF-κB [71]. In Balb/c mice, treatment either topically or systemically with NF-κB decoy oligonucleotides followed by exposure to UVB light has been shown to significantly reduce the UV induced cutaneous swelling, epidermal hyperplasia and secretion of the proinflammatory cytokines IL-1β, TNFα and IL-6 [72].

There is also strong evidence suggesting that NF-κB plays an important role in carcinogenesis. In a malignant melanoma cell line an enhanced nuclear localisation of p50/p65 was seen compared to normal human epidermal melanocytes [73].

NF-κB activation has also been suggested to play a role in some autoimmune diseases [74] as well as being a part of the pathogenesis in the infection with B. burgdorfei also called Lyme disease [75].

Nuclear receptors

Only selected nuclear receptors will be discussed here (( figure 2 )).

PPAR

In the hyperproliferative psoriatic epidermis the expression of PPARδ is significantly upregulated compared to un-involved psoriatic skin whereas the expression of PPARα and PPARγ are more controversial [78, 80]. Furthermore several of the hydroxylated eicosanoids that accumulate in psoriatic lesions are potent PPARδ agonists.

Activation of PPARα has been shown to decrease the inflammatory response in both an irritant and an allergic contact dermatitis animal model [81]. Furthermore PPARα and PPARδ but not PPARγ have been suggested to play a role in wound healing [82].

Another interesting finding demonstrating the complexity of signal transduction is that both PPARα, PPARγ and PPARδ have been shown to be involved in the modulation of inflammatory responses via negative cross-talk with transcription factors such as NF-κB and AP-1 [78, 83].

RXR and VDR

RA can modulate the response of keratinocytes to mitogens and is a pleiotropic regulator of epidermal differentiation. RA has both stimulatory and inhibitory effects on keratinocyte differentiation. These diverse effects are mediated through binding to specific subtypes of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) which leads to activation of different sets of responsive genes.

Vitamin D₃ is another important nuclear hormone ligand that regulates keratinocyte homeostasis [85] by decreasing cell proliferation and inducing differentiation. Vitamin D₃ binds to its specific nuclear receptor, the Vitamin D₃ receptor (VDR). The VDR/ligand complex can then dimerize with the thyroid receptor (TR), the RXR, the RAR or another VDR but the biologically most important complex is the VDR/RXR heterodimer allowing for cross-talk between these two signaling pathways.

The complexity of the interplay between VDR, RAR and RXR has recently been reviewed by Barsony and Prufer [86].

Conclusion

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