The effect of the glucocorticoid dexamethasone on peroxisome proliferation: a defined physiological cell culture model to study

Fig. 4.3: Overview of the different extra- and intracellular stimuli and their effect on peroxisome dynamics.

Note that induction of cytosolic or mitochondrial (Mito) oxidative stress does not affect peroxisome dynamics, while UV irradiation, low cell density, growth factor and PUFA stimulation leads to a tubulation of peroxisomes. To facilitate this, membrane receptors and intracellular (damage) signalling might contribute to exert a peroxisomal response.

Dexamethasone (Dexa) treatment induces peroxisome elongation which is linked to the regulation of Pex11β while 6-OHDA addition did not affect peroxisomal, but mitochondrial dynamics. Note that tubulation of peroxisomes might indicate peroxisomal growth and division to increase peroxisome numbers or mediate a metabolic function by e.g.

increasing the surface-to-volume ratio.

4.3.3 The effect of the glucocorticoid dexamethasone on peroxisome

desirable which allows to reliably monitor alterations of peroxisome dynamics after application of a defined, well-characterized stimulus.

In this study, we took advantage of an AR42J cell culture model that is usually employed to study the differentiation of pancreatic acinar cells and the biogenesis of zymogen granules (Swarovsky et al., 1988; Faust et al., 2008; Borta et al., 2010). Stimulation of AR42J cells which are derived from rat exocrine pancreatic tumour cells, with glucocorticoids such as dexamethasone was shown to lead to the coupled induction of exocrine proteins and a profound remodelling of intracellular compartments involved in the secretory pathway (Logsdon et al., 1985; Logsdon et al., 1987; Swarovsky et al., 1988). For instance, secretory differentiation markers such as chymotrypsinogen and amylase show a profound induction in regard to their mRNA and synthesis rates and the formation of zymogen granules is induced (Scheele, 1993). The AR42J cell culture model has been established in our lab (Faust et al., 2008; Borta et al., 2010) and was thus used here to study the effects of dexamethasone, a synthetic fluorinated glucocorticoid, on peroxisome dynamics. AR42J cells were treated with either 10 nM dexamethasone, a concentration used to stimulate differentiation of AR42J cells, or 1 µM of the compound, representing a more pathophysiological concentration (Du et al., 2009). Induction was carried out for 6-72 hours with fresh dexamethasone and peroxisome morphology as well as induction of chymotrypsinogen (a zymogen granule marker) was assessed by epifluorescence microscopy. As described before, a significant increase of zymogen granules/chymotrypsinogen was observed (Fig. 3.32 C), verifying successful dexamethasone action, but interestingly dexamethasone treatment also resulted in a significant, dose-dependent tubulation of peroxisomes (Fig. 3.31; 3.32; 4.3). Untreated controls displayed the pattern of peroxisome morphology after seeding as described for mammalian cells (Schrader et al., 1994; Schrader et al., 1996a), i.e. an increase in tubular forms 24 hours after seeding (and here 6 hours after induction) with a subsequent increase in spherical forms reminiscent of fission. In contrast to that application of dexamethasone provided a time- and dose-dependent stimulus for continuous peroxisome elongation (Fig.

3.32 B). Although induction of tubular peroxisomes mirrored the kinetics of zymogen granule formation, it is important to note that not all cells with tubular peroxisomes also displayed granular structures, insinuating that both processes are not necessarily coupled. Interestingly, also singular addition of dexamethasone and subsequent removal of the stimulus was sufficient to induce peroxisome tubulation, even 48 hours after removal of dexamethasone (Fig. 3.33). As the observed peroxisome morphology after dexamethasone treatment largely mimicked the morphology of peroxisomes observed after overexpression of Pex11pβ (Fig.

3.34), further analysis aimed at investigating changes in the induction profile of the three rat Pex11 isoforms by a semi-quantitative PCR approach (SQ-PCR) (Fig. 3.35). AR42J cells were stimulated with dexamethasone (10 nM and 1 µM) for 24, 48 and 72 hours and the induction of Pex11α, Pex11β and Pex11γ was assessed in comparison to the housekeeping gene GAPDH by SQ-PCR. The peroxisomal key enzyme AOX was included as a control for peroxisome proliferation. Compared to the stable expression of GAPDH, AOX was clearly induced after dexamethasone treatment. This induction pattern was mirrored at the protein level (Fig. 3.31) and is indicative of peroxisome proliferation. Pex11α, the inducible isoform (Abe et al., 1998), was similarly expressed and was only clearly detected after dexamethasone induction, mirroring the pattern of AOX induction. In contrast to that, clear signals corresponding to Pex11β were seen in controls at every time point investigated, in line with its constitutive expression in all tissues. However, Pex11β was also induced 24 and 48 hours after treatment with dexamethasone when compared to controls (Fig. 3.31). The Pex11pβ antibody failed to recognize the rat protein upon immunoblotting, thus induction of Pex11pβ was not verified on the protein level. Nonetheless, as the induction of AOX on the protein level followed the pattern observed on the mRNA level (Fig. 3.31), the observations made by SQPCR were reliable. Comparison between Pex11β levels in control cells at different time points revealed it to be induced after 72 hours (96 hours after seeding) when compared to the 24 and 48 hour expression levels. This might be due to a potential “normal” induction of Pex11β in the course of cell growth and differentiation. While Pex11α and Pex11β were clearly induced by dexamethasone addition, Pex11γ signals remained barely detectable throughout the experiment and were thus concluded to play a minor role in regulating dexamethasone-induced alterations of peroxisome morphology. Altogether, the rat AR42J cell line was established as a valuable cell culture model to study alterations of peroxisome dynamics and their regulation. In contrast to other stimuli, e.g. serum addition, the initial signal leading to peroxisome elongation was corresponded to one well-defined external stimulus, the glucocorticoid dexamethasone. The results obtained here were also highly reproducible. Moreover, dexamethasone addition was directly linked to the activation of key components of the peroxisomal growth and division machinery, Pex11α and Pex11β.

However, as peroxisome tubulation is initiated upon expression of Pex11pβ and Pex11pγ, but not Pex11pα (Fig. 3.34), the peroxisomal phenotype of dexamethasone addition is most

peroxisome dynamics was identified in this study. Dexamethasone-induced peroxisomal elongation thus provides an easy, amenable and highly reproducible cell culture model to assess changes in the expression profile of peroxisomal genes involved in the regulation of peroxisome dynamics by e.g. large scale expression profiling. It might also contribute to the identification of novel components regulating peroxisome dynamics and offers a more straightforward approach to study regulation of peroxisome abundance when compared to fibrate-induced peroxisome proliferation in rodents.

The molecular action of glucocorticoids has so far been linked to several mechanisms:

glucocorticoid activities can be divided into genomic effects, mediated by the cytosolic glucocorticoid receptor alpha (cGCR) and other non-genomic effects (Pratt, 1998; Almawi &

Melemedjian, 2002; Adcock & Lane, 2003; Wikstrom, 2003; Buttgereit et al., 2004;

Buttgereit et al., 2005; Stahn et al., 2007). In the classical mode of action, the unligated cGCR, a member of the steroid hormone receptor family, resides in the cytoplasm as a multiprotein complex including heat shock proteins, immunophilins, chaperones (such as Src) and several kinases of the MAPK family (Pratt, 1998; Almawi & Melemedjian, 2002;

Wikstrom, 2003). The cGCR itself contains 3 key domains, the N-terminal transactivation domain, a DNA-binding zinc finger domain and a ligand binding domain (Wikstrom, 2003).

Glucocorticoids can pass the plasma membrane due to their lipophilic structure and are able to bind the receptor which in turn leads to dissociation of the cCGR complex, homo-dimerization of the receptor and its translocation into the nucleus occurs where it binds specific DNA binding sites (glucocorticoid response elements, GREs) (Almawi &

Melemedjian, 2002). In line with their use as inflammatory drugs, GREs induce anti-inflammatory proteins, but also genes for gluconeogenesis. In addition to this positive regulation of GREs (“transactivation”), there is also negative regulation (“transrepression”) of negative GREs, inhibiting e.g. transcription of interleukin-1 (Falkenstein et al., 2000).

Moreover, apart from direct GRE binding, the activated GC/GCR complex can also interact with other transcription factors or compete with other nuclear co-activators, leading to a transrepression of genes (Reily et al., 2006; Stahn et al., 2007) In terms of the genomic effects of cGCR action, the activation, translocation and binding to GREs takes about 30 minutes, while changes on the cellular or tissue level become apparent after hours or days (Stahn et al., 2007). Other non-genomic effects are thought to occur by a direct effect of the lipophilic GCs on biological membranes (e.g. by intercalation with the mitochondrial membrane) (Buttgereit

& Scheffold, 2002; Buttgereit et al., 2004), downstream effects of the release of the cGCR complex via e.g. release of MAPK and activation of a potential membrane-bound GCR

(Gametchu et al., 1999; Croxtall et al., 2000; Buttgereit et al., 2004). Up until now, there is only limited information on the role of glucocorticoid action on peroxisome function and/or dynamics. However, dexamethasone was shown to regulate the expression of PPARα in rat hepatocytes (Rao & Subbarao, 1997; Plant et al., 1998; Lawrence et al., 2001). Most of these studies were carried out in rat hepatocytes to not only determine the contribution of glucocorticoids to peroxisome proliferation, but also to determine the basis of the cell proliferation and tumour growth observed in rodents upon feeding with peroxisome proliferators. It was also demonstrated that the addition of glucocorticoids during culturing of primary rat hepatocytes was necessary to maintain PPARα expression and thus responsiveness to peroxisome proliferators (Mitchell et al., 1984). Upon addition of peroxisome proliferators, electron microscopy and morphometric analysis then revealed in increase in the fractional volume of peroxisomes. However, dexamethasone application alone was not sufficient to induce the expression of the peroxisome marker PMP70 in mice (Lawrence et al., 2001). Our observations on the induction of Pex11α and Pex11β indicate a genomic effect of dexamethasone on peroxisome dynamics; however, the exact molecular regulation requires further analysis. The induction of AOX and Pex11α, both of which are regulated by PPARα, might indicate that dexamethasone induces PPARα which in turn activates transcription of its targets. To analyze a potential contribution of PPARα stimulation to the observed phenotype, AR42J cells were treated with bezafibrate, a potent inducer of PPARα-mediated peroxisome proliferation in rat hepatocytes. It was surmised that if dexamethasone effects stem from indirect consequences of PPARα activation, the observed morphology and induction pattern in AR42J cells would be similar. However, no effect on peroxisome elongation or proliferation was observed (Fig. 3.37 A-C). Furthermore, no induction of AOX or Pex11α – PPARα target genes – was detected (Fig. 3.37 D), indicating AR42J cells of rodent pancreatic origin only respond weakly to bezafibrate, unlike rat hepatocytes. Similar results were obtained using ETYA, another peroxisome proliferating drug (data not shown). This might be due to a low expression of PPARα in AR42J cells which actually contain features of both exocrine and endocrine pancreas. In line with this, though primarily endocrine pancreas was investigated, PPARα expression was indicated to be low in pancreatic tissue, while the glucocorticoid receptor was moderately expressed (Bookout et al., 2006). As our findings indicate AR42J cells to be low responders to bezafibrate and thus the

activate the downstream signal transduction onto the molecular and then peroxisomal level.

The maintained peroxisome elongation after glucocorticoid stimulation was not indicative of growth and division processes upon which peroxisomal tubules subsequently would be divided. Interestingly, elongation of the peroxisomal membrane and formation of complex structures have been linked to metabolic processes such lipid synthesis or penicillin production (Kollatakudy et al., 1987; Kabeya et al., 2005; Kiel et al., 2005b) and were suggested to facilitate a metabolic function of peroxisomes by generating a uniform biochemical distribution of proteins, increasing the surface to volume ratio and exchanging metabolites (Schrader & Fahimi, 2006). Thus, continuous membrane elongation after dexamethasone stimulation might represent a specific peroxisome morphology indicative of a metabolic function instead of an growth and division process. As glucocorticoids have vast anti-inflammatory capacities (Buttgereit et al., 2004; Stahn et al., 2007), a shift of peroxisomal dynamics to a more tubulated structure might modulate the anti-inflammatory response, by e.g. a stimulation peroxisomal breakdown of the inflammatory mediators leukotriene or prostaglandin as well as arachidonic acid (AA). In line with this, application of AA also induced elongated peroxisomes (Schrader et al., 1998a).

Thus, rat pancreatic AR42J cells have a high potential to serve as a model system for dexamethasone-induced peroxisomal elongation (Fig. 4.3). It provides an amenable system to easily assess changes in the expression profile of peroxisomal genes involved in the regulation of peroxisome dynamics after application of a well-defined stimulus. Future studies using large scale expression profiling in this system might contribute to the identification of novel components regulating peroxisome morphology and dynamics.