METODOLOGÍA utilizada en LA ELABORACIÓN DEL PET [10]

PGE2 exerts a number o f effects at the cellular level. These include the regulation o f proliferation, extracellular matrix synthesis, immune and inflammatory responses. Some o f these effects will be discussed in the following section with particular emphasis on fibroblast responses to PGE2.

1.4.9,1 Regulation o f proliferation

PGE2 can stimulate or inhibit proliferation depending on the cell type and culture conditions. For instance, PGE2 is mitogenic for NIH3T3 cells but is a potent

inhibitor o f lung fibroblast and smooth muscle cell proliferation (Watanabe et al

1996; McAnulty et al., 1997; Belvisi et al., 1998). The following section will focus on the inhibitory effects of PGE2 on proliferation with particular reference to lung fibroblasts. Mechanisms for the anti-proliferative effects o f PGE2 will also be considered.

PGE2 is a potent inhibitor of lung fibroblast proliferation (Elias et aL, 1985; Bitterman et al., 1986; Jordana et al, 1987; Fine and Goldstein 1987; Klein and Adamson 1989; Oliver et al., 1989; McAnulty et a l, 1997). Both exogenously administered PGE2 (Fine and Goldstein 1987; Oliver et al., 1989; McAnulty et al., 1997) and endogenous PGE2 synthesis by alveolar macrophages (Elias et al., 1985; Bitterman et al., 1986; Jordana et al., 1987) and type II epithelial cells (Klein and Adamson 1989) inhibits the proliferation o f lung fibroblasts. In addition, treatment of fibroblasts with growth factors such as IL-1(3, TN F-a and TGF-p 1.3 results in inhibition of lung fibroblast proliferation due to autocrine synthesis o f PGE2 (Elias et al., 1988; McAnulty et al., 1997). In the case o f TGF- pi-3, this was demonstrated in foetal lung fibroblasts (McAnulty et al., 1997) and it is not known whether PGE2 mediates the anti-proliferative effects of TGF-pi.3 in fibroblasts derived from adult lung. Furthermore, in diseases such as pulmonary fibrosis, fibroblast hyperplasia occurs and the contribution of inhibitory pathways in circumventing this response has received little attention.

The mechanism for PGE2-mediated growth inhibition is not well known but evidence suggests a role for cAMP. PGE2 elevates levels o f intracellular cAMP in human lung fibroblasts (Baum et al., 1980; Clark et al., 1982; 1983) suggesting the activation o f the EP2 and/or EP4 receptors. The cAMP analogue, dibutryl cAMP also blocks the proliferation of lung fibroblasts in response to serum (Fine and Goldstein 1987). A functional cAMP response element has been localized to the promoter region o f COX-2 (Wadleigh and Herschman 1999) and in monocytic cells and mast cells this response element regulates COX-2 gene expression (Inoue

et al., 1994; Reddy et al., 2000). An interaction between CRE and PGE2-mediated growth inhibition has not been investigated. However, a role for cAMP in PGE2- mediated inhibition o f proliferation has been demonstrated in arterial smooth

muscle cells and in hepatic stellate cells (Bomfeldt et aL, 1997; Mallat et aL,

1998). Figure 1.3 shows a potential model for the regulation o f proliferation by PGE2 in lung fibroblasts stimulated with TGF-p 1. In fibroblasts, low concentrations o f TGF-p] induce autocrine synthesis o f PDGF, which mediates the mitogenic effects of TGF-pi (Battegay et aL, 1990). Both PDGF and high concentrations o f TGF-pi induce PGE2 release (Habenicht et aL, 1985; McAnulty et aL, 1997). This response is most likely mediated by COX-2 since in airway smooth muscle cells, inhibition of cell growth by PGE2 is associated with COX-2 expression (Belvisi et aL, 1998). At this point activation of the cAMP pathway has been demonstrated in smooth muscle cells and hepatic stellate cells (Bomfeldt

et aL, 1997; Mallat et aL, 1998). This implicates the EP2 and/or EP4 receptor pathway. This cascade of events would ultimately result in the inhibition of fibroblast proliferation.

In liver fibrosis, hepatic stellate cells differentiate to an activated myofibroblast phenotype, exhibiting enhanced mitogenicity (Friedman 2000). However, treatment o f normal stellate cells with either PDGF or thrombin results in growth inhibition via a PGE2/CAMP signal which counteracts the promitogenic effects conferred by these growth factors (Mallet et aL, 1998). The authors suggest that a shift in the balance between positive and negative signals for cell proliferation contributes to the proliferation of myofibroblasts in liver fibrosis. A similar scenario may explain the hyperproliferation o f lung fibroblasts in pulmonary fibrosis although this type of interaction between mediators has not been previously investigated in lung fibroblasts.

1.4,9.2 Regulation o f collagen synthesis

PGE2 suppresses collagen production in human fibroblast cell lines derived from the heart and lungs (Goldstein and Polgar 1982; Fine et aL, 1989; Saltzman et aL,

PDGF TGF-pi E P J E P , cAMP PLAJPLC COX-2 DNA s y n th e s is / Proliferation fibroblast

Figure 1.3 Potential model for regulation of fibroblast proliferation by PG E2

Following stimulation with TGF-p|, both growth stimulatory (blue arrows) and inhibitory signals (red arrows) are generated. The mitogenic effects o f TGF-p, are ascribed to autocrine synthesis o f PDGF (Battegay et aL, 1990). However, both PDGF and TGF-p, induce PGE2 synthesis (Habenicht et a l , 1985;

McAnulty et a l , 1997). The inhibitory effects o f PGE2 are likely to be mediated via the EP] or EP4

Exogenous PGE2 downregulates total collagen production (Goldstein and Polgar 1982; Saltzman et aL, 1982; Brilla et a i, 1995) by decreasing levels of collagen gene expression (Fine et aL, 1989) and promoting degradation o f newly synthesized collagen (Brilla et aL, 1995).

PGE2 also depresses collagen production induced by mediators such as IL -la (Goldring and Krane 1987), bradykinin (Goldstein and Polgar 1982) and TGF-Pj (Fine et al, 1989; McAnulty et aL, 1995). In addition to these effects, PGE2 downregulates basal and TGF-p 1-induced expression o f lysyl oxidase (LO) mRNA (Roy et aL, 1996), an enzyme involved in the post-translational modification of collagen. Recently, PGE2 released from bronchial epithelial cells was shown to inhibit fibroblast-mediated collagen gel contraction (Mio et aL, 1998). Subsequently glucocorticoids were shown to augment fibroblast contractility due to inhibition o f endogenous PGE2 synthesis (Skold et aL, 1999). Thus in the absence o f PGE2, and in the presence of fibroblast stimulatory mediators, the potential exists for dysregulated collagen production.

The mechanism for PGE2-mediated suppression of collagen synthesis is not well known, however, as with PGE2’s effects on cell proliferation, studies suggest a role for cAMP. Intracellular cAMP levels increase after incubation o f lung fibroblasts with PGE2 (Saltzman et aL, 1982). Likewise, PGEi also inhibits collagen production and increases intracellular degradation (Barile et aL, 1988) with a concomitant increase in levels o f cAMP (Baum et aL, 1980). Furthermore, the suppression o f basal, IL-lp and TGF-p 1-induced LO mRNA expression is transduced via the EP2/CAMP pathway in lung fibroblasts (Choung et aL, 1998).

PGE2 is the major eicosanoid product of fibroblasts and occurs at high concentrations in normal lung where it promotes extracellular matrix homeostasis by limiting fibroblast proliferation and collagen synthesis. During inflammatory episodes, fibroblast activity increases and the following section focuses on the interaction between PGE2 and pro-inflammatory mediators.