Índice de Tablas
VALIDACIÓN DE LA METODOLOGIA
ERK in kératinocytes in vitro by retroviral infection with an activated M E K l construct does increase the proliferative rate of the cells (Haase et al., 2001) but without affecting the proportions of stem and transit amplifying cells in the population (Zhu et al., 1999). Here I show that constitutive ERK activation delays although does not block suspension induced differentiation. This effect could explain the increase in keratinocyte proliferation as a delay in onset of terminal differentiation may allow transit amplifying cells to divide a greater num ber of tim es before w ithdraw ing from the cell cycle and expressing differentiation markers. This is an attractive explanation as the transit am plifying compartment is thought to be expanded in psoriatic lesions (lizuka et al., 1996). My results, together with those of Ingo Haase (Haase et al., 2001) agree with the emerging role of ERK as a key regulator of both keratinocyte proliferation and differentiation (Cai et al., 2002; Dajee et al., 2002; Haase and Hunzelmann, 2002; Zhu et al., 1999) (see Chapter 1) and lend credence to the hypothesis that increased/disregulated ER K activity could contribute to the pathogenesis of hyperproliferative epidermal lesions such as psoriasis.
Transgenic mice expressing |3l integrins from the involucrin prom oter generate sporadic psoriatic-like lesions (Carroll et al., 1995), leading to the conclusion that the suprabasal (31 integrin present in hyperproliferative epidermis may have a causal role in the generation of the lesion. Suprabasal |3l integrin correlated with activated ERK in psoriasis and in hyperproliferative lesions from Involucrin-|3l integrin mice, suggesting that these integrins are signalling to ERK which in turn is responsible for the hyperproliferation and abnormal differentiation of kératinocytes present in the lesion. H ow ever, phenotypically normal epidermis from Involucrin-Pl integrin mice did not display active ERK although it retains transgene expression which implies that if the suprabasal p i integrin is able to signal to
ERK, it is doing so in a non-constitutive fashion. It has been reported that kératinocytes derived from In v o lu crin -p l integrin m ice, when placed in suspension to induce differentiation and transgene expression and then plated onto antibodies specific for the transgenic integrin, are able to activate E R K l/2 (Haase et ah, 2001), thus demonstrating that the suprabasal integrins are capable of transducing a signal. However, the lack of accumulation of major ligands for p i integrin in the hyperproliferative lesions (laminin 1, type IV collagen and fibronectin) (Carroll et ah, 1995) would mean that the integrins would therefore not be activated and so directly signal to ERK. The possibility that suprabasal integrins are ligated should not be com pletely discounted though as, for example, a novel type XIII collagen is found in all layers of human epidermis (Peltonen et ah, 1999).
I show here a possible indirect mechanism of ERK activation by suprabasal integrins. Kératinocytes derived from Involucrin-a2pi integrin mice produce more I L - la compared to controls. I L - la , in turn, is able to activate ERK in kératinocytes. This indirect m echanism could provide an explanation as to the non-constitutive nature of ERK activation by suprabasal p i integrins as the response of kératinocytes to IL-1 is tightly regulated. Kératinocytes express high levels of an intracellular variant of the IL -lra (Corradi et ah, 1995; Haskill et ah, 1991), which blocks access of IL-1 to the IL -lR l (Arend, 1993). Furthermore, kératinocytes can be induced to express the decoy receptor for IL-1, IL-1R2 (Groves et ah, 1995a), which titrates out active IL-1 without transducing a signal into the cell (Mantovani et ah, 2001). Dermal fibroblasts can also act as a sink for keratinocyte-derived IL-1, probably through receptor binding and internalisation and would thus influence the accumulation of IL-1 in the epidermis (Boxman et ah, 1996).
Chapter 3. ERK M APK activation by integrins in hyperproliferative epidermis
Injection of I L - la into human skin potently induces an inflammatory response (Camp et al., 1990; Dowd et al., 1988; Groves et al., 1992) and overexpression of I L - l a from the K eratin 14 p ro m o ter in the ep id erm is o f tra n sg e n ic m ice g e n erate s a macrophage/monocyte dermal infiltrate (Groves et al., 1995b). Thus, an increased I L - la production from the kératinocytes of Involucrin-|3l integrin transgenic mice could explain the inflam m atory infiltrate observed in these mice. Furtherm ore, double-paracrine stim ulation pathw ays betw een kératinocytes and derm al fib ro b lasts have been characterised where keratinocyte-derived IL -I induces fibroblasts to produce KGF, GM- CSF and IL-6 which can subsequently regulate keratinocyte pro liferatio n and differentiation (Boxman et al., 1996; Maas-Szabowski et al., 1999; Maas-Szabowski et al., 2000; Szabowski et al., 2000). Therefore, elevated I L - l a production from Involucrin- a 2 p i integrin kératinocytes could trigger the increased production/accumulation of many different cytokines and growth factors from both an accompanying inflammatory infiltrate and dermal fibroblast stimulation. Many of these could also contribute to activation of ERK in the kératinocytes.
A basic w orking hypothesis o f m echanism s in v o lv ed in the g eneration of hyperproliferative and inflam m atory epiderm al lesions via suprabasal p i integrin expression is depicted in Fig. 3.4. This model, based on the evidence presented in this chapter, led to further studies in the next two chapters where I address the exact role of ERK in driving epiderm al hyperproliferation, d ifferentiatio n abnorm alities and inflammation (Chapter 4) and further, the exact mechanism s by which suprabasal p i integrin expression elevates I L - la production (Chapter 5).