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Cell Biology International (2010) 34, 543–552 (Printed in Great Britain)
Z-FA.FMK activates duodenal epithelial cell proliferation through oxidative stress, NF-κB and IL-1β in d-GalN/TNF-α-administered mice
Selda Gezginci‑Oktayoglu*1, Sehnaz Bolkent*, Bertan Boran Bayrak† and Refiye Yanardag†
*Istanbul University, Faculty of Science, Department of Biology, 34134-Vezneciler, Istanbul, Turkey, and †Istanbul University, Faculty of Engineering, Department of Chemistry, 34320-Avcilar, Istanbul/Turkey

This study was designed to evaluate the effect of Z-FA.FMK (benzyloxycarbonyl-l-phenylalanyl-alanine-fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z-FA.FMK, the third group received d-GalN (d-galactosamine) and TNF-α (tumour necrosis factor-α) and the fourth group was given both d-GalN/TNF-α and Z-FA.FMK. When d-GalN/TNF-α was administered alone, we observed an increase in IL-1β-positive and active NF-κB-positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)-positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z-FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d-GalN/TNF-α injection. On the contrary, the Z-FA.FMK pretreatment before d-GalN/TNF-α blocked these effects. Based on these findings, we suggest that Z-FA.FMK might act as a proliferative mediator which is controlled by IL-1β through NF-κB and oxidative stress in duodenal epithelial cells of d-GalN/TNF-α-administered mice.

Key words: d-GalN/TNF-α, duodenal epithelial cell, IL-1β, NF-κB, oxidative stress, Z-FA.FMK

Abbreviations: CAT, catalase, DETAE, Experimental Medical Research and Application Institute, d-GalN, d-galactosamine, ECM, extracellular matrix, GSH, glutathione, IEC, intestinal epithelial cell, IL-1β, interleukin-1β, i.p., intraperitoneal, i.v., intravenous, LI, labelling index, LPO, lipid peroxidation, Masson, Masson’s trichrome, NF-κB, nuclear factor κB, IκB, inhibitor of NF-κB, PAS, Periodic Acid Schiff, PCC, protein carbonyl content, PCNA, proliferative cell nuclear antigen, SBP, spontaneous bacterial peritonitis, SOD, superoxide dismutase, TNF, tumour necrosis factor, Z-FA.FMK, benzyloxycarbonyl-l-phenylalanyl-alanine-fluoromethylketone

1To whom correspondence should be addressed (email

1. Introduction

The gastrointestinal mucosa is continuously exposed to microorganisms and other luminal antigens, which activate the intestinal mucosa immune response. The lymphocytes and other immunological cells present in the mucosa interact with each other and also with intestinal epithelial cells through the release of cytokines (Kagnoff, 1993). The intestinal epithelial cells also synthesize cytokines and their receptors, which indicates that they serve as an early signalling system to the immune and inflammatory cells of the host (Kagnoff, 1993; Stadnyk, 1994). Cytokines are produced locally, exert their autocrine, paracrine and endocrine actions (Stadnyk, 1994) and participate in the development of an inflammatory cascade (Kelso, 1989).

The intestinal epithelium is a continuously renewable population of cells arising from the proliferative stem cells located within the crypts. Renewal of the surface epithelium is required to maintain its functional integrity. Certain cytokines are potent stimulators of lymphocyte cell proliferation (Kelso, 1989) and affect cell proliferation also in different cell types such as cancer cells (Wu et al., 1993). IL-1β (interleukin-1β) is a cytokine that induces proliferation in numerous cell types (Dinarello, 1996). As for the intestine, previous reports have indicated that IL-1β directly induces the proliferation of cultured intestinal smooth muscle cells (Owens and Grisham, 1993). These findings strongly suggest a role of IL-1β in inflammation-induced intestinal cell proliferation; however, limited data are available regarding the effects of IL-1β on cytokine-induced proliferation of duodenal mucosa epithelial cells.

A central feature of TNF (tumour necrosis factor) signalling in multiple cell types is the induction of apoptosis, which typically involves the binding of the cytokine to its receptor, followed by the recruitment of adapter molecules and activation of caspase enzymes. In addition to the activation of the mitochondrial pathway of apoptosis, TNF can also mobilize proteases from the lysosome (Jaattela, 2004). In particular, cathepsin B has been implicated as an effector protease in the TNF cascade of cell death (Foghsgaard et al., 2001). This protease can induce cell demise by stimulating mitochondrial permeabilization (Guicciardi et al., 2000). The same TNF receptor can also recruit the adapter protein TNF receptor-associated factor 2, which has recently been shown to drive NF-κB (nuclear factor kappa B) translocation to the nucleus and the induction of antiapoptotic and proinflammatory genes (Yamaguchi et al., 2009).

Lysosomal cathepsins play important roles in many physiological and pathological processes in diverse cell types in tissues (Brix et al., 2008). Cysteine cathepsin B is mainly expressed in endocytic compartments, and is an important mediator of protein processing and catabolism in the mucosa cells of the intestine (Mayer et al., 2006). Besides their principle function in protein degradation of internalized proteins within endolysosomes, cathepsins are also involved in the processing of precursor proteins to biologically active peptides in endosomes of enteroendocrine cells (Furuhashi et al., 1991). Excessive degradation of ECM (extracellular matrix) components in tissue can result in destruction of the basal lamina, which may induce epithelial regeneration (Basson, 2001). It is well-established that a misbalance of proteases and their inhibitors can contribute to pathological conditions of the gastrointestinal tract during inflammation of the intestine (Medina and Radomski, 2006).

Peptidyl fluoromethylketones with amino acids phenylalanine and alanine in the P1 and P2 positions, respectively, have been shown to be irreversible inhibitors of some members of the cathepsin enzyme family (Rasnick, 1985; Ahmed et al., 1992). In particular, Z-FA.FMK (benzyloxycarbonyl-l-phenylalanyl-alanine-fluoromethylketone) was found to be a potent inactivator of the human cathepsin B and binds tightly to the active site of the enzyme. Once bound to the active site, the fluoromethylketone group alkylates the cysteine residue in the active site and forms a covalent bond, which irreversibly blocks its proteolytic activity (Lawrence et al., 2006). Z-FA.FMK was also found to inhibit LPS (lipopolysaccharide)-induced cytokine production in macrophages by blocking the transactivation potential of NF-κB (Schotte et al., 2001). These data suggest that cathepsin B may be a good target for therapeutic intervention for the treatment of inflammatory intestinal diseases using Z-FA.FMK.

The transcription factor NF-κB regulates expression of genes involved in immune responses, inflammation, cell survival and cell proliferation. In its active DNA-binding form, NF-κB is a dimer, composed of members of the Rel family. Five mammalian proteins of this family are known: p65, RelB, c-Rel, NF-κB1 (p-50 and its precursor p105) and NF-κB2 (p52 and its precursor p100). The NF-κB dimers are primarily retained in the cytoplasm by association with IκB (inhibitor of NF-κB). Phosphorylation and degradation of IκB releases NF-κB and enables its translocation to the nucleus, where it regulates inflammatory transcriptional programs (Hoffmann and Baltimore, 2006). Proinflammatory cytokines, such as TNF-α and IL-1, allow rapid nuclear translocation of NF-κB (Baeuerle and Henkel, 1994). Thus, NF-κB is a key mediator of TNF-α responses and an attractive target for therapeutic intervention against inflammation and inflammatory diseases.

Oxygen-free radicals are molecules produced continuously in cells by several mechanisms. The generation of oxygen-free radicals is physiological. In most circumstances, oxygen-free radicals are neutralized immediately by enzymatic scavengers. But, when formation of oxygen free radicals overwhelms radical neutralization in cells, oxidative stress occurs. As they are very reactive, they react with all biological substances such as proteins, polysaccharides and nucleic acids, resulting in tissue injury (Dröge, 2003). It has been suggested that oxygen-free radicals are responsible for a wide variety of diseases or conditions (Keshavarzian et al., 1990). Furthermore, it was proposed that diverse agents such as IL-1 and TNF-α activated NF-κB by causing oxidative stress (Bowie and O’Neil, 2000).

Besides participating in cell immunity, TNF-α also plays an important role in many diseases such as hepatic failure (Gezginci and Bolkent, 2007) and inflammatory bowel disease (Song et al., 2004). Patients with severe hepatitis are at high risk of serious complications such as SBP (spontaneous bacterial peritonitis), which is an important cause of death in patients with severe hepatitis (Franca et al., 2001). SBP is related to the changes of intestinal permeability and bacterial translocation (Chiva et al., 2003), as well as the production of inflammatory mediators such as TNF and IL (Rodriguez-Ramos et al., 2001). However, additional studies are needed to clarify the regulation mechanism of intestinal mucosa damage by hepatic failure. Therefore in this study, we have performed a series of experiments on intestines of mice that have hepatic failure caused by d-GalN (d-galactosamine)/TNF-α as shown previously (Gezginci and Bolkent, 2007; Gezginci-Oktayoglu et al., 2008).

2. Materials and methods

2.1. Animals

Male BALB/c mice (2.5–3 months old, weighing ≈25 g) were obtained from the Istanbul University Experimental Medical Research and Application Institute (DETAE). All experiments were carried out in accordance with the guidelines of the Animal Care and Use Committee of DETAE (permission number 14 and permission date 24th February 2004). The mice were maintained at a constant temperature of 22±1°C with 12-h light/dark cycles and fed a standard pellet chow, ad libitum. The animals were fasted for 15 h prior to treatments, but were allowed free access to water.

2.2. Experimental design

The animals were divided into four experimental groups as follows: Group 1 (n = 10) – animals receiving i.v. (intravenous) injections of physiological saline; Group 2 (n = 6) – administered 8 mg/kg Z-FA.FMK in 10% dimethyl sulphoxide by i.v. injection; Group 3 (n = 10) – mice treated with 100 mg/kg d-GalN and 15 μg/kg TNF-α by sequential i.p. (intraperitoneal) injections; Group 4 (n = 7) – treated with 100 mg/kg d-GalN and 15 μg/kg TNF-α by sequential i.p. injection 1 h after administration of 8 mg/kg Z-FA.FMK. Groups 1 and 3 were killed after 4 h by ether anaesthesia, while the mice in Groups 2 and 4 were killed after 5 h; 1 h for the effect of Z-FA.FMK and 4 h to induce injury. The intestine tissues were immediately taken up for microscopic and biochemical investigations. Recombinant TNF-α was obtained from Calbiochem. d-GalN was purchased from Acros Organics and Z-FA.FMK from Sigma Chemical Co.

2.3. Histology

The intestine tissues were cut into small pieces and fixed in Bouin’s solution. Following dehydration in an ascending series of ethanol concentrations, the samples were cleared in xylene and embedded in paraffin. Slides were stained with Masson’s trichrome (Masson) and PAS (Periodic Acid Schiff). All sections were examined with an Olympus CX-45 light microscope and photographed using an Olympus DP71 digital camera.

2.4. Immunohistochemistry

For the immunohistochemical stainings, mouse polyclonal antibody against PCNA (proliferative cell nuclear antigen, NeoMarkers MS-106-P; dilution 1:50; 30 min at room temperature), rabbit polyclonal antibody against IL-1β (Santa Cruz Biotechnology sc-7884; dilution 1:100; overnight at 4°C) and goat polyclonal antibody against NF-κB p50 (Santa Cruz Biotechnology sc-114G; dilution: 1:50; 2 h at room temperature) were used. Slides were placed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven for IL-1β labelling. The endogenous peroxidase activity was eliminated by treatment with 3% hydrogen peroxide. Later, a Histostain Plus Broad Spectrum Kit (Zymed 85-9043) was employed. Sections were incubated with blocking solution, and the primary antibodies were applied. They were stained consecutively with biotinylated secondary antibody and streptavidin peroxidase conjugate. Peroxidase activity was demonstrated by staining with a freshly prepared substrate solution of 3-amino-9-ethylcarbazole. Sections were counterstained with Mayer haematoxylin and mounted in glycerol vinyl alcohol. For quantification of immunostaining, 10 fields (0.0506 mm2) were randomly selected for counting. The immunopositive duodenal epithelial cell percentage [LI% (labelling index)] was calculated as follows:

2.5. Biochemical analysis

For biochemical analysis, tissue samples of the intestine were washed in saline and kept frozen until the day of the experiments. The tissue samples in cold saline were homogenized in a glass apparatus to make a 10% (w/v) homogenate. After centrifugation, the supernatant fraction was removed for determination. The markers of oxidative stress and antioxidant activity levels of tissue LPO (lipid peroxidation), GSH (glutathione) and PCC (protein carbonyl content), as well as SOD (superoxide dismutase) and CAT (catalase) activities were assessed. The LPO levels were determined by the method of Ledwozyw et al. (1986), the PCC was assessed as described by Levine et al. (1990), the GSH levels were measured by Beutler’s method (1975), CAT activity was determined according to the method of Aebi (1984), SOD activity was assessed by the method described by Mylroie et al. (1986), and total protein levels were measured by the method of Lowry et al. (1951). Furthermore, tissue collagen content was measured as a fibrosis marker. Evaluation of collagen content was made according to the method published by Lopez-De Leon and Rojkind (1985).

2.6. Statistical analysis

Statistical analysis for immunohistochemical data were conducted using the GraphPad Prism software version 4.00 (GraphPad Prism). Data were subjected to one-way ANOVA (analysis of variance) with Tukey post-test and results are expressed as means±S.E.M. The biochemical results were evaluated by unpaired t test and ANOVA using the NCSS statistical computer package and are reported as means±S.D.

3. Results

3.1. Z-FA.FMK prevents formation of the morphological changes in d-GalN/TNF-α-induced duodenal injury

Saline or Z-FA.FMK-injected control mouse duodenum had normal histological apperance, while d-GalN/TNF-α-treated mice had moderate histopathological changes. We observed mononuclear cell infiltraton, hyperaemia, oedema inside the expanded and compressed villi, and a decrease in the PAS-positive reaction in the goblet cells and brush border in the duodenum of these mice. On the other hand, Z-FA.MK injection blocked d-GalN/TNF-α-induced histopathogical changes in the duodenum except for compression of the villi (Figure 1).

3.2. Z-FA.FMK blocks NF-κB activation in epithelial cells of d-GalN/TNF-α-injected mouse duodenum

To determine if NF-κB activation might be occurring in the duodenum of mice treated with d-GalN/TNF-α, samples were analysed immunohistochemically for nuclear translocation of NF-κB p50. Consequently, cytoplasmic NF-κB immunolabelling was considered inactive, while nuclear or nuclear and cytoplasmic NF-κB were indicative of active NF-κB. Z-FA.FMK pretreatment did not produce a significant change in the number of inactive (8.7% in saline-injected and 4.7% in Z-FA.FMK-treated mice, P>0.05) or active NF-κB+ duodenal epithelial cell index (1.8% in saline-injected or Z-FA.FMK-treated mice, P>0.05). Increased NF-κB+ duodenal epithelial cell index was observed in a nuclear distribution by d-GalN/TNF-α injection (25.5%), compared with a small amount of active NF-κB+ cells in control tissue samples (P<0.001). Moreover, Z-FA.FMK blocks the nuclear translocation of NF-κB p50 in duodenal epithelial cells of d-GalN/TNF-α-treated mice (1%, P<0.001) (Figure 2).

3.3. Z-FA.FMK blocks IL-1β expression in epithelial cells of d-GalN/TNF-α-injected mouse duodenum

We could not detect IL-1β+ duodenal epithelial cells in control groups, while IL-1β was present in the interstitial areas. On the other hand, d-GalN/TNF-α injection triggered IL-1β synthesis in these cells (53.1%, P<0.001) and interstitial areas (2.2%, P>0.05). Z-FA.FMK pretreatment of d-GalN/TNF-α-injected mice prevented increase in the number of IL-1β+ duodenal epithelial cells (0.6%, P<0.001) and increase in interstitial areas (0.8%, P<0.05) (Figure 3).

3.4. Z-FA.FMK induces duodenal epithelial cell proliferation in d-GalN/TNF-α-injected mice

Duodenal epithelial cell proliferation was examined by PCNA immunolabelling. Z-FA.FMK injection decreased PCNA+ duodenal epithelial cell number significantly (43.4% in saline-injected and 27.6% in Z-FA.FMK-treated mice, P<0.01). Duodenal epithelial cell proliferation was also significantly decreased in d-GalN/TNF-α-injected mice (14.2%, P<0.001). On the other hand, the Z-FA.FMK pretreatment prevented decrease in the number of PCNA+ duodenal epithelial cell (39.8%, P<0.001) (Figure 4).

3.5. Z-FA.FMK prevents d-GalN/TNF-α-induced oxidative stress in intestine

Intestinal GSH level (P<0.001) and CAT or SOD activities (P<0.05) were significantly decreased by d-GalN/TNF-α administration. Pretreatment with Z-FA.FMK prevented this reduction in GSH levels (P<0.01) and CAT or SOD activities (P<0.05) in the intestines of these mice (Table 1). On the other hand, intestinal LPO and PCC levels increased significantly with d-GalN/TNF-α administration compared with saline-injected mice (P<0.001). Pretreatment with Z-FA.FMK reduced intestinal LPO (P<0.05) and PCC (P<0.001) levels in these mice. Z-FA.FMK pretreatment to control animals also increased LPO and PCC levels in comparison to saline-injected mice (Table 1).

Table 1 GSH, CAT and SOD activities, LPO levels, PCC and collagen content in control and experimental groups of mice

Values are means±S.D. n = 10 animals for each group.

Measurement Saline Z-FA.FMK d-GalN/TNF Z-FA.FMK+d-GalN/TNF
GSH (nmol of GSH/mg of protein) 20.15±1.49 14.76±2.92 11.16±0.60* 13.58±0.89
CAT (units/mg of protein) 1.15±0.16 0.60±0.09 1.00±0.11∥ 1.21±0.04
SOD (units/mg of protein) 1.55±0.15 2.46±0.27 1.22±0.11¶ 1.75±0.37
LPO (nmol of MDA/mg of protein) 1.28±0.08 1.72±0.10 2.03±0.21† 1.50±0.01
PCC (nmol of carbonyl/mg of protein) 1.09±0.12 1.45±0.25 2.53±0.36‡ 1.70±0.19
Collagen (μg/mg of protein) 15.88±1.33 15.62±1.91 22.36±2.27§ 15.76±1.20

,,,§Pt test<0.001 compared with saline.

,Pt test<0.05 compared with saline.

3.6. Z-FA.FMK prevents d-GalN/TNF-α-induced fibrosis in intestine

Intestinal collagen levels increased by d-GalN/TNF-α administration compared with saline-injected mice (P<0.001). Pretreatment with Z-FA.FMK reduced intestinal collagen levels (P<0.001) in these mice (Table 1).

5. Discussion

Great interest has been focused on the possible involvement of inflammatory cytokines in intestinal disease. It is evident that IL-1β and TNF-α are involved in the pathogenesis of inflammatory bowel disease (Reimund et al., 1996). Experiments conducted on mice have shown that treatment with IL-1 induced intestinal pathology (Mowat et al., 1993). It has also been demonstrated that administration of IL-1 receptor agonist reduced intestinal inflammation and injury in ulcerative colitis (Mansfield et al., 1994). However, there are no data in the literature on the possible role of IL-1β in the pathogenesis of intestine caused by d-GalN/TNF-α and its control mechanism. The results presented in this study showed an increase in IL-1β synthesis, nuclear translocation of NF-κB, and oxidative stress and a decrease in proliferation of duodenal epithelial cells with d-GalN/TNF-α administration. Moreover, cathepsin B inhibition by Z-FA.FMK prevented all of these changes in d-GalN/TNF-α-administered mouse intestine. These findings suggest that cathepsin B is involved in d-GalN/TNF-α-inhibited duodenal epithelial cell proliferation regulated by oxidative stress, NF-κB and IL-1β in mice.

Cathepsins are expressed ubiquitously by various cell types including intestinal cells (Mayer et al., 2006). Saftig et al. (1995) have observed that cathepsin d-deficient mice showed a progressive atrophy of intestinal mucosa, suggesting that it is essential for regulation of IEC (intestinal epithelial cell) proliferation. The results reported in this study present new data about the possible role of cathepsin B in duodenal epithelial cell proliferation. We observed that pretreatment with Z-FA.FMK, a pharmacological cathepsin B inhibitor, to d-GalN/TNF-α-administered mice caused stimulation of duodenal epithelial cell proliferation. This finding indicates an inhibitor effect of cathepsin B on duodenal epithelial cell proliferation in d-GalN/TNF-α-administered mice just like on hepatocytes (Gezginci and Bolkent, 2007) and pneumocytes (Oztay et al., 2009). On the other hand, we observed that cathepsin B was an activator protein in kidney tubular epithelial cell proliferation in the same model (Gezginci-Oktayoglu et al., 2008). Hence, the proliferator effect of cathepsin B can be conceived as cell type-specific.

It is well known that oxidative stress and cathepsin B are involved in the mediation of TNF-α signalling (Gezginci and Bolkent, 2007; Gezginci-Oktayoglu et al., 2008; Oztay et al., 2009). A direct action of ROS (reactive oxygen species) on lysosomes may increase the lysosomal membrane permeabilization. To support this hypothesis, rat liver lysosomes were permeabilized upon exposure to a hydroxyl radical generating system (Olsson et al., 1989). Furthermore, it has been observed that H2O2 caused lysosomal membrane permeabilization in a tissue culture system (Zhao et al., 2001). In additon, Windelborn and Lipton (2008) have proposed that superoxide stimulates cathepsin B release by increasing lysosomal membrane permeabilization in neurons. However, duodenal cells have not been tested in this regard, and it is unknown if there is a link between oxidative stress and cathepsin B in TNF-α-induced duodenal injury. We have observed that cathepsin B inhibition prevented d-GalN/TNF-α-induced oxidative stress in the duodenum. This finding suggested a possible cathepsin B-mediated oxidative stress formation mechanism in duodenum.

It is thought that NF-κB is associated with the expression of various cytokines in inflammation. NF-κB has been reported to exhibit increased expression in nuclear extracts from local mucosal tissue specimens of patients with inflammatory bowel disease (Schreiner et al., 1998). Reports have been published to evaluate the inflammation-suppressing effect of antisense oligonucleotide for NF-κB on mice with ulcerative colitis (Murano et al., 2000) or ischaemia–reperfusion injury of the small intestine (Mallick et al., 2005). The role of NF-κB subunit p65 in preventing TNF-α-induced injury has been well studied (Alcamo et al., 2001). However, there has been little evidence to support a role of p50 in protecting from TNF. Gadjeva et al. (2007) reported that the absence of p50 is the primary factor that sensitizes mice to TNF-α-induced apoptosis of enterocytes. In agreement with this study, we could detect barely any apoptotic duodenal epithelial cells by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) labelling in D-GalN/TNF-α-injected mouse intestine (data not shown), while there was an increase in NF-κB p50 subunit expressed in duodenal epithelial cells. We have also detected numerous proliferative duodenal epithelial cells in these mice. Hence, the data reported here underline that p50 subunit might present as an antiapoptotic and proliferative instead of an apoptotic mediator in our model.

An important query in the field of NF-κB research is about its activation mechanism. A model for explaining this question proposed a relationship between NF-κB activation and oxidative stress (Schreck et al., 1992). This hypothesis was based on some evidence such as the observation that direct addition of H2O2 to culture medium activates NF-κB in some cell lines (Schreck et al., 1991; Meyer et al., 1993). Ultimately, this theory proposed H2O2 as the central second messenger of NF-κB activation (Schmidt et al., 1995). We have observed an increase in NF-κB+ duodenal epithelial cell number, LPO and PCC levels as indicators of oxidative stress, and a decrease in GSH level and SOD activity as indicators of a lack of antioxidant defence, while there was no change in CAT activity with d-GalN/TNF-α injection. Because SOD is a well-known antioxidant enzyme that scavenges superoxide radicals, while CAT ameloriates H2O2, our observations showed superoxide radical instead of H2O2 as the central messenger of NF-κB activation. However, decreased GSH level in d-GalN/TNF-α-treated mice suggested a possible role of H2O2 in NF-κB activation. It is possible that H2O2 and superoxide radical are present in the activation of transcription factor NF-κB. Hence, our findings brought a new standpoint in the oxidative stress-regulated NF-κB activation hypothesis.

In vital organs such as the intestine tissue, injury caused by a range of insults including oxidative stress can result in the development of progressive fibrosis, leading to ultimate organ failure (Franklin, 1997). In our study, collagen contents of intestine tissue were significantly increased at the same time as oxidative stress markers with d-GalN/TNF-α injection. These findings indicated the presence of oxidative stress-induced fibrotic activity in d-GalN/TNF-α-administered mice. On the other hand, Z-FA.FMK pretreatment attenuated the fibrotic activity together with oxidative damage. All of these data suggested a possible mechanism for cathepsin B-related oxidative stress induction in TNF-α-mediated intestinal injury as in the other organs such as liver, kidney and lung (Gezginci and Bolkent, 2007; Gezginci-Oktayoglu et al., 2008; Oztay et al., 2009). While IL-1β production by IEC in vitro has been reported (Lundqvist et al., 1996), it does not occur in vivo. For example, McCall et al. (1994) have reported that IEC did not produce IL-1β in the chronically inflamed human intestine. We also could detect barely any IL-1β+ duodenal epithelial cells in control mice in our study. To understand the mechanisms underlying IL-1β production, Waterhouse and Stadnyk (1999) detached IEC, and they saw that following detachment, these cells rapidly expressed IL-1β mRNA. This finding indicates a possible relationship between ECM degradation and IL-1β production in IEC. So, it is also possible that ECM-degrading proteases, such as cathepsins (Wolf et al., 2003), have a role in triggering IL-1β production in these cells. To check this possibility, we inhibited cathepsin B by using Z-FA.FMK. Consequently, cathepsin B inhibition blocked IL-1β expression in duodenal epithelial cells of d-GalN/TNF-α-administered mice. On the other hand, the mice treated with d-GalN/TNF-α had numerous IL-1β+ duodenal epithelial cells. These findings indicate an important activator role of cathepsin B in IL-1β production in duodenal epithelial cells.

It has been suggested that IL-1 has a role as a mediator of cellular proliferation in intestinal mucosa. Zachrisson et al. (2001) have found that administration of IL-1β or TNF-α stimulated DNA synthesis in small intestine cell lines. However, another study has shown that IL-1β activated epithelial cell restitution after mucosal injury but had no effect on cell proliferation (Dignass and Podolsky, 1993). Vignolini et al. (1998) have demonstrated that the increase in IL-1β expression was associated with a decrease in intestinal cell proliferation. In addition, Ohama et al. (2007) have shown that IL-1β inhibited intestinal smooth muscle cell proliferation in an organ culture system. In agreement with the last-mentioned results, our data showed an inhibitory role of IL-1β on duodenal epithelial cell proliferation in d-GalN/TNF-α-induced intestinal injury, since the increase in IL-1β synthesis is associated with a decrease in duodenal epithelial cell proliferation.

To the best of our knowledge, this is the first study to demonstrate the proliferative effect of Z-FA.FMK on duodenal mucosal epithelial cells under the control of oxidative stress, NF-κB and IL-1β in d-GalN/TNF-α-administered mice. In summary, TNF-α can cause oxidative stress in duodenal epithelial cells. Oxidative stress activates nuclear translocation of NF-κB, which results in IL-1β expression. Finally, IL-1β inhibits duodenal epithelial cell proliferation according to our findings. On the other hand, in the presence of Z-FA.FMK all of these TNF-α-mediated molecular events are inhibited. So, Z-FA.FMK might be used as a regenerative agent in TNF-α-mediated intestinal damage.

Author contribution

Selda Gezginol-Oktayoglu and Sehnez Bolkent were responsible for data collection and analysis, microscopic evaluations, and the design and writing of the manuscript. Berton Boran Bayrak and Refiye Yanardag were responsible for the biochemical analysis and the writing of the manuscript.


The authors would like to thank MedSanTek® for providing the IL-1β antibody.


This work was supported by the Research Fund of Istanbul University [project number BYP-3886].


Aebi, H (1984) Catalase in vitro. Methods Enzymol 105, 121-6
Medline   1st Citation  

Ahmed, NK, Martin, LA, Watts, LM, Palmer, J, Thornburg, L, Prior, J and Esser, RE (1992) Peptidyl fluoromethyl ketones as inhibitors of cathepsin B: implication for treatment of rheumatoid arthritis. Biochem Pharmacol 44, 1201-7
Crossref   Medline   1st Citation  

Alcamo, E, Mizgerd, JP, Horwitz, BH, Bronson, R, Beg, AA and Scott, M (2001) Targeted mutation of TNF-receptor I rescues the RelA-deficient mouse and reveals critical role for NF-kappa B in leukocyte recruitment. J Immunol 167, 1592-600
Medline   1st Citation  

Basson, MD (2001) In vitro evidence for matrix regulation of intestinal epithelial biology during mucosal healing. Life Sci 69, 3005-18
Crossref   Medline   1st Citation  

Baeuerle, PA and Henkel, T (1994) Function and activation of NF-κB in the immune system. Annu Rev Immunol 12, 141-79
Medline   1st Citation  

Beutler, E (1975) Glutathione in red cell metabolism. A mannual of biochemical methods. Grune and Stratton, New York
1st Citation  

Bowie, A and O’Neil, LAJ (2000) Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 59, 13-23
Crossref   Medline   1st Citation  

Brix, K, Dunkhorst, A, Mayer, K and Jordans, S (2008) Cysteine cathepsins: cellular roadmap to different functions. Biochime 90, 194-207
Crossref   1st Citation  

Chiva, M, Guarner, C, Peralta, C, Llovet, T, Gomez, G, Soriano, G and Balanzo, J (2003) Intestinal mucosal oxidative damage and bacterial translocation in cirrhotic rats. Eur J Gastroenterol Hepatol 15, 145-50
Crossref   Medline   1st Citation  

Dignass, AU and Podolsky, DK (1993) Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor beta. Gastroenterol 105, 1323-32
1st Citation  

Dinarello, CA (1996) Biologic basis for interleukin-1 in disease. Blood 87, 2095-147
Medline   1st Citation  

Dröge, W (2003) Oxidative stress and aging. Adv Exp Med Biol 543, 191-200
Medline   1st Citation  

Foghsgaard, L, Wissing, D, Mauch, D, Lademann, U, Bastholm, L, Boes, M, Elling, F, Leist, M and Jäättelä, M (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153, 999-1010
Crossref   Medline   1st Citation  

Franca, AV, De Souza, JB, Silva, CM and Soares, EC (2001) Long-term prognosis of cirrhosis after spontaneous bacterial peritonitis treated with ceftriaxone. J Clin Gastroenterol 33, 295-8
Crossref   Medline   1st Citation  

Franklin, TJ (1997) Therapeutic approaches to organ fibrosis. Int J Biochem Cell Biol 29, 79-89
Crossref   Medline   1st Citation  

Furuhashi, M, Nakahara, A, Fukutomi, H, Kominami, E, Grube, D and Uchiyama, Y (1991) Immunocytochemical localization of cathepsins B, H, and L in rat gastroduodenal mucosa. Histochem 95, 231-9
1st Citation  

Gadjeva, M, Wang, Y and Horwitz, BH (2007) NF-κB p50 and p65 subunits control intestinal homeostasis. Eur J Immunol 37, 2509-17
Crossref   Medline   1st Citation  

Gezginci, S and Bolkent, S (2007) The effect of Z-FA.FMK on d-galactosamine/TNF-alpha-induced liver injury in mice. Cell Biochem Funct 25, 277-86
Crossref   Medline   1st Citation   2nd   3rd   4th   5th  

Gezginci-Oktayoglu, S, Tunali, S, Yanardag, R and Bolkent, S (2008) Effects of Z-FA.FMK on d-Galactosamine/tumor necrosis factor-alpha-induced kidney injury and oxidative stress in mice. Mol Cell Biochem 309, 9-20
Crossref   Medline   1st Citation   2nd   3rd   4th  

Guicciardi, ME, Deussing, J, Miyoshi, H, Bronk, SF, Svingen, PA, Peters, C, Kaufmann, SH and Gores, GJ (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106, 1127-37
Crossref   Medline   1st Citation  

Hoffmann, A and Baltimore, D (2006) Circuitry of nuclear factor kappaB signaling. Immunol Rev 210, 171-86
Crossref   Medline   1st Citation  

Jaattela, M (2004) Multiple cell death pathways as regulators of tumour initiation and progression. Oncogene 23, 2746-56
Crossref   Medline   1st Citation  

Kagnoff, MF (1993) Immunology of the intestinal tract. Gastroenterol 105, 1275-80
1st Citation   2nd  

Kelso, A (1989) Cytokines: structure, function and synthesis. Curr Opin Immunol 2, 215-25
Crossref   Medline   1st Citation   2nd  

Keshavarzian, A, Morgan, G, Sedghi, S, Gordon, JH and Doria, M (1990) Role of reactive oxygen metabolites in experimental colitis. Gut 31, 786-90
Crossref   Medline   1st Citation  

Lawrence, CP, Kadioglu, A, Yang, AL, Coward, WR and Chow, SC (2006) The cathepsin B inhibitor, Z-FA-FMK, inhibits human T cell proliferation in vitro and modulates host response to pneumococcal infection in vivo. J Immunol 177, 3827-36
Medline   1st Citation  

Ledwozyw, A, Michalak, J, Stepien, A and Kadziolka, A (1986) The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 155, 275-83
Crossref   Medline   1st Citation  

Levine, RL, Garland, D, Oliver, CN, Amici, A, Climent, I, Lenz, AG, Ahn, BW, Shaltiel, S and Stadtman, ER (1990) Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186, 464-78
Medline   1st Citation  

Lopez-De Leon, A and Rojkind, MJ (1985) A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. Histochem Cytochem 33, 737-43
1st Citation  

Lowry, OH, Rosebrough, NJ, Farr, AL and Randall, RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-75
Medline   1st Citation  

Lundqvist, C, Melgar, S, Yeung, MM, Hammarström, S and Hammarström, ML (1996) Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions. J. Immunol 157, 1926-34
Medline   1st Citation  

Mallick, IH, Yang, WX, Winslet, MC and Seifalian, AM (2005) Pyrrolidine dithiocarbamate reduces ischemia–reperfusion injury of the small intestine. World J Gastroenterol 11, 7308-13
Medline   1st Citation  

Mansfield, JC, Holden, H, Tarlow, JK, Di Giovine, FS, McDowell, TL, Wilson, AG, Holdsworth, CD and Duff, GW (1994) Novel genetic association between ulcerative colitis and the anti-inflammatory cytokine interleukin-1 receptor antagonist. Gastroenterol 106, 637-42
1st Citation  

Mayer, K, Schwartz, S, Lentze, MJ, Kalff, JC and Brix, K (2006) Extracellular localization of intestinal cathepsins: implications of their actions during post-operative ileus. In XLI Congress of the European Society for Surgical Research (Vollmar B, ed.), Medimond International Proceedings, Rostock, Germany
1st Citation   2nd  

McCall, RD, Haskill, S, Zimmermann, EM, Lund, PK, Thompson, RC and Sartor, RB (1994) Tissue interleukin 1 and interleukin-1 receptor antagonist expression in enterocolitis in resistant and susceptible rats. Gastroenterol 106, 960-72
1st Citation  

Medina, C and Radomski, MW (2006) Role of matrix metalloproteinases in intestinal inflammation. J Pharmacol Exp Ther 318, 933-8
Crossref   Medline   1st Citation  

Meyer, M, Schreck, R and Baeuerle, PA (1993) H2O2 and antioxidants have opposite effects on activation of NF-κB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J 12, 2005-15
Medline   1st Citation  

Mowat, AM, Hutton, AK, Garside, P and Steel, M (1993) A role for interleukin-1α in immunologically mediated intestinal pathology. Immunol 80, 110-1
1st Citation  

Murano, M, Maemura, K, Hirata, I, Toshina, K, Nishikawa, T, Hamamoto, N, Sasaki, S, Saitoh, O and Katsu, K (2000) Therapeutic effect intracolonically administered nuclear factor κB (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp Immunol 120, 51-8
Crossref   Medline   1st Citation  

Mylroie, AA, Collins, H, Umbles, C and Kyle, J (1986) Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol Appl Pharmacol 82, 512-20
Crossref   Medline   1st Citation  

Ohama, T, Hori, M, Momotani, E, Elorza, M, Gerthoffer, WT and Ozaki, H (2007) IL-1β inhibited intestinal smooth muscle proliferation in an organ culture system: involvement of COX-2 and iNOS induction in musculares resident machrophages. Am J Physiol Gastrointest Liver Physiol 292, G1315-22
Medline   1st Citation  

Olsson, GM, Svensson, I, Zdolsek, JM and Brunk, UT (1989) Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction. Virchows Arch B Cell Pathol Incl Mol Pathol 56, 385-91
Crossref   Medline   1st Citation  

Owens, MW and Grisham, MB (1993) Cytokines increase proliferation of human intestinal smooth muscle cells: possible role in inflammaion induced structure formation. Inflammation 17, 481-7
Crossref   Medline   1st Citation  

Oztay, F, Gezginci-Oktayoglu, S, Bayrak, BB, Yanardag, R and Bolkent, S (2010) Cathepsin B inhibition improves lung injury associated to d-galactosamine/tumor necrosis factor-alpha-induced liver injury in mice. Mol Cell Biochem 333, 65-72
Crossref   Medline   1st Citation   2nd   3rd  

Rasnick, D (1985) Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B. Anal Biochem. 149, 461-5
Medline   1st Citation  

Reimund, JM, Wittersheim, C, Dumont, S, Muller, CD, Kenney, JS, Baumann, R, Poindron, P and Duclos, B (1996) Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn’s disease. Gut 39, 684-9
Crossref   Medline   1st Citation  

Rodriguez-Ramos, C, Galan, F, Diaz, F, Elvira, J, Martin-Herrera, L and Giron-Gonzalez, JA (2001) Expression of proinflammatory cytokines and their inhibitors during the course of spontaneous bacterial peritonitis. Dig Dis Sci 46, 1668-76
Crossref   Medline   1st Citation  

Saftig, P, Hetman, M, Schmahl, W, Weber, K, Heine, L, Massmann, H, Koster, A, Hess, B, Evers, M and Von Figura, K (1995) Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells. EMBO J 14, 3599-608
Medline   1st Citation  

Schmidt, KN, Amstad, P, Cerutti, P and Beauerle, PA (1995) The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol. 2, 13-22
Medline   1st Citation  

Schotte, P, Schauvliege, R, Janssens, S and Beyaert, R (2001) The cathepsin B inhibitor z-FA.fmk inhibits cytokine production in macrophages stimulated by lipopolysaccharide. J Biol Chem 276, 21153-21157
Crossref   Medline   1st Citation  

Schreck, R, Meier, M, Mannel, DN, Droge, W and Baeuerle, PA (1992) Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med 175, 1181-94
Crossref   Medline   1st Citation  

Schreck, R, Rieber, P and Baeuerle, PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10, 2247-58
Medline   1st Citation  

Schreiner, S, Nikolaus, S and Hampe, J (1998) Activation of nuclear factor kappa B inflammatory bowel disease. Gut 42, 477-84
Medline   1st Citation  

Song, HL, Lu, S, Ma, L, Li, Y and Liu, P (2004) Effect of TNF on tight junctions between the epithelial cells of the intestinal mucosa barrier. Shijie Huaren Xiaohua Zazhi 12, 1303-6
1st Citation  

Stadnyk, AW (1994) Cytokine production by epithelial cells. FASEB J 8, 1041-7
Medline   1st Citation   2nd  

Vignolini, F, Nobili, F and Mengheri, E (1998) Involvement of interleukin-1β in zinc deficiency-induced intestinal damage and beneficial effect of cyclosporine A. Life Sci. 62, 131-41
Medline   1st Citation  

Waterhouse, CCM and Stadnyk, AW (1999) Rapid expresion of IL-1β by epithelial cells in vitro. Cell Immunol 193, 1-8
Crossref   Medline   1st Citation  

Windelborn, JA and Lipton, P (2008) Lysosomal release of cathepsins causes ischemic damage in the rat hippocampal slice and depends on NMDA-mediated calcium influx, arachidonic acid metabolism, and free radical production. J Neurochem 106, 56-69
Crossref   Medline   1st Citation  

Wolf, K, Mazo, I, Leung, H, Engelke, K, Von Andrian, UH, Deryugina, EI, Strongin, AY, Bröcker, EB and Friedl, P (2003) Compensation mechanism in tumor cell migration mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160, 267-77
Crossref   Medline   1st Citation  

Wu, S, Boyer, CM, Whitaker, RS, Berchuck, A, Wiener, JR, Weinberg, JB and Bast, RC (1993) Tumor necrosis factor alpha as an autocrine and paracrine growth factor for ovarian cancer: monokine induction of tumor cell proliferation and tumor necrosis factor alpha expression. Cancer Res 53, 1939-44
Medline   1st Citation  

Yamaguchi, T, Miyashita, C, Koyano, S, Kanda, H, Yoshioka, K, Shiba, T, Takamatsu, N and Ito, M (2009) JNK-binding protein 1 regulates NF-kappaB activation through TRAF2 and TAK1. Cell Biol Int 33, 364-8
Crossref   Medline   1st Citation  

Zachrisson, K, Neopikhanov, V, Samali, A and Uribe, A (2001) Interleukin-1, interleukin-8, tumor necrosis factor alpha and interferon gamma stimulate DNA synthesis but have no effect on apoptosis in small-intestinal cell lines. Eur J Gastronterol Hepatol 13, 551-59
Crossref   1st Citation  

Zhao, M, Brunk, UT and Eaton, JW (2001) Delayed oxidant-induced cell death involves activation of phospholipase A2. FEBS Lett 509, 399-404
Crossref   Medline   1st Citation  

Received 19 December 2009/27 January 2010; accepted 3 February 2010

Published as Cell Biology International Immediate Publication 3 February 2010, doi:10.1042/CBI20090485

© The Author(s) Journal compilation © 2010 Portland Press Limited

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)