The results reported here are in line with and extend our previous results (Coconnier et al., 1998; 2000). We demonstrate that the L. monocytogenes thiol-activated exotoxin LLO promotes the mucin exocytosis in a human mucin- and PGs-secreting cell line. In parallel, we report for the first time that LLO induced the upregulation of several MUC genes encoding for membrane-bound mucins. When examining the mechanism by which LLO induced this cellular response, we found that LLO did not activate the nuclear transcriptional factors NF-κB and AP-1.
LLO enhanced specifically the mucin exocytosis in PGs- and mucin-secreting human polarized intestinal HT29-MTX cells
Host cell heparan sulphate PGs (HSPG) are integral components of plasma membranes, ubiquitously distributed among cell populations of mammalian tissues (for a review, see Prydz and Dalen, 2000). As for mucins, regulated and constitutive secretion pathways of PGs have been characterized (Brion et al., 1992). Interestingly, it has been established that PGs acted as receptors for a large variety of virulent Gram-negative and Gram-positive bacteria (for a review, see Rostand and Esko, 1997), particularly in L. monocytogenes pathogenicity (Alvarez-Dominguez et al., 1997; Jonquieres et al., 2001). Results reported here clearly outlined that the HMGs secreted upon LLO stimulation are in majority of a mucin nature.
A high level of MUC5AC mucin was found in culture medium of LLO-stimulated HT29-MTX cells. It is important to note that physiologically, the MUC5AC is known to be expressed in the respiratory tract, gastric mucosa and reproductive mucosa. The high expression level of MUC5AC mucin in the human adenocarcinoma colonic HT29-MTX and HT29-Cl.16E cell lines is reminiscent of the differentiation of the human fetal colonic epithelium (for a review, see Zweibaum et al., 1991). It could be not excluded that other mucins such as intestinal mucins expressed by the cells are secreted upon LLO stimulation. However, the levels of those mucins could not be determined by radioimmunoassay, because antibodies directed against these mucins are not currently suitable for quantitative determination.
Surprisingly, increase in MUC5AC mucin secretion in LLO-stimulated HT29-MTX cells develops without upregulation of the corresponding MUC5AC gene. There are two secretory pathways in mucin-secreting polarized cells (for reviews, Forstner, 1995; Laboisse et al., 1995). One is a steady vesicular constitutive pathway of mucin exocytosis in which small vesicles could be continuously transported directly to the cell surface to undergo exocytosis of their mucin content. Mucins are also stored in large vesicles to form a granule mass which undergoes the so-called regulated pathway of mucin exocytosis. This mucin exocytosis pathway is regulated by specific stimuli involving signalling molecules. We have previously demonstrated that LLO-induced mucin exocytosis is insensitive to pharmacological blockers of neuroendocrine receptors and coupled signalling systems, and to inflammatory agents known to be involved in controlling the regulated pathway of mucin exocytosis (Coconnier et al., 1998). Moreover, on the basis of the observation that LLO-stimulated mucin exocytosis is dependent on microtubular organization (Coconnier et al., 2000), a characteristic of the constitutive vesicular pathway of mucin exocytosis (for a review, see Forstner, 1995), we have previously suggested that LLO may activate baseline mucin secretion. Results reported here indicate that MUC5AC mucin is one of the mucins transported and secreted through the vesicular pathway of mucin exocytosis stimulated by LLO. In the related HT29–18 N2 clone, all conventional large secretory granules have been shown stain with MUC5AC antibodies (Stanley and Phillips, 1999). The fact that the MUC5AC mucin secretion was increased upon LLO stimulation without upregulation of the corresponding MUC5AC gene indicates that the mucin is from a preformed origin. We have conducted an additional experiment in which mucins have been radiolabelled during a short pulse of 1 h and 2 h (data not shown). No increase in mucins exocytosis upon LLO stimulation was observed after a short pulse compared with the increased mucins exocytosis found after a 18 h of pulse. In consequence, this result disagrees with our previous hypothesis that LLO stimulate the constitutive, non-regulated pathway of mucins exocytosis ant in contrast suggests that the mucins liberated upon LLO stimulation are not from a newly synthesized origin but from a preformed origin. Interestingly, Forstner (1995) in discussing the properties and characteristics of the mucin secretion pathways in intestinal cells, has postulated that a population of small vesicles could be stored as lateral granules on the granule mass formed in majority by large granules, which is involved in the regulated pathway of mucin exocytosis. We have previously reported that the large mucin-containing granules, located beneath the apical surface of the mucin-secreting cells and characteristic of the regulated pathway of mucin exocytosis, remain unchanged in LLO-stimulated cells (Coconnier et al., 2000). In order to reconcile our previous (Coconnier et al., 1998; 2000) and the present results, one explanation is that LLO could stimulate a subpopulation of vesicles containing MUC5AC in the granule mass forming the storage of mucins in intestinal cells.
LLO elicits upregulation of MUC genes encoding for membrane-bound mucins without activation of the nuclear transcription factors NF-κΒ and AP-1
Epithelial mucins can be classified into two main groups: membrane-associated and secreted mucins. The secreted mucins can be subdivided in two groups: gel-forming mucins and non-gel-forming mucins. Currently, 12 human mucin genes (MUC1-4, MUC5B, MUC5AC, MUC6-7, MUC11, MUC12, MUC13, and MUC14) have been identified (for reviews, Gendler and Spicer, 1995;Seregni et al., 1997; Williams et al., 1999a; Yin and Lloyd, 2001). More than one mucin gene can be expressed in a given tissue and in cultured mucin-secreting cell lines. The complete primary amino acid sequence of only MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B and MUC7 have been deduced (for a review, see Crawley et al., 1999). The secreted gel-forming mucins are encoded by a cluster of four mucin genes (MUC2, MUC6, MUC5B and MUC5AC) located on the chromosom 11p15 (Desseyn et al., 1997). Four genes,MUC1, MUC3, MUC4 and MUC12, encoding for membrane-associated mucins have been characterized. The MUC1 gene is located on chromosome 1q21–24, MUC2, MUC6, MUC4 on 3q29, MUC7 on 4q13–21, and MUC3, MUC11 and MUC12 on 7q22 (Gum et al., 1997; Williams et al., 1999a, b).
We reported here the expression of MUC1-4, MUC5B, MUC5AC, MUC11 and MUC12 genes, but not of MUC6 and MUC7 genes in HT29-MTX cells. The observation that MUC1-4, MUC5B and MUC5AC genes are expressed in HT29-MTX cells is consistent with previous reports (Lesuffleur et al., 1993; Debailleul et al., 1998). We observed that among the MUC genes investigated, LLO promotes specifically the upregulation of MUC3, MUC4 and MUC12 genes. The present work is the four report that describes MUC gene upregulation upon bacterial infection. Indeed, it has been recently reported that the Gram-negative P. aeruginosa and Haemophilus influenzae and Gram-positive Staphylococcus aureus, S. epidermis and Streptococcus pyogenes promoted the upregulation of MUC2 and MUC5AC genes in NCIH292 epithelial cells (Dohrman et al., 1998; Li et al., 1998; Wang et al., 2002). All the MUC genes found to be upregulated by LLO encoded for membrane-associated mucins (Nollet et al., 1998; Crawley et al., 1999; Moniaux et al., 1999; Williams et al., 1999b).
Many cellular responses involving inducible genes are regulated by the widely used transcription nuclear factors NF-κB and AP-1 (May and Ghosh, 1999). The pleiotropic mediator of induction and tissue-specific gene control NF-κB is a member of the Rel family of transcriptional activator proteins. In activated cells, degradation of IκB proteins allows the entry of free NF-κB into the nucleus, where it binds to its target sequences and activates transcription. AP-1 is a homo- and heterodimeric transcription factor composed of members of the Jun and Fos family of DNA-binding proteins (Angel and Karin, 1991). Several stimuli such as growth factors, cytokines, hormones and microbial infections are known to induce this transcription factor. Exoproducts of P. aeruginosa promoted upregulation of MUC2 gene in NCIH292 and HM3 epithelial cells, through the activation of the transcription factor NF-κB via a Src-dependent Ras-MEK1/2-ERK1/2-pp90rsk pathway (Li et al., 1998). When examining whether or not the transcription factors NF-κB and AP-1 are involved in LLO-induced upregulation of MUC3, MUC4 and MUC12 genes, we found that upregulation occurs without activation of NF-κB and c-Fos synthesis. Cellular responses through the nuclear translocation of NF-κB by L. monocytogenes virulence factors have been recently documented, but our results are not in agreement with those showing that LLO elicits cellular responses through a NF-κB-dependent mechanism. Interestingly, we observed that control infection of the HT29-MTX cells by S. enterica serovar Typhimurium and L. monocytogenes EGD is followed by degradation of IκB proteins. LLO and L. monocytogenes in the absence of cell invasion elicits NF-κB-dependent cellular responses (Kayal et al., 1999; Rose et al., 2001). However other reports suggest the possibility that the L. monocytogenes virulence factors synergistically act to promote NF-κB-dependent cellular responses in several cell types (Schwarzer et al., 1998; Hauf et al., 1997). Analysing the NF-κB activation mechanism, it appears that the cell response involves two phases of activation, a transient activation phase by lipoteichoic acid (LTA) followed by a second persistent activation phase by the two phospholipases encoded by the virulence genes plcA and plcB. Our results obtained with L. monocytogenes EGD infection and LLO stimulation are consistent with the hypothesis that L. monocytogenes virulence factors synergistically act to promote NF-κB-dependent cellular responses. Indeed, we observed that IκB protein degradation develops when the HT29-MTX cells are infected with L. monocytogenes EGD, whereas LLO is not able to promote this effect.
Pathophysiological consequences of the LLO-induced upregulation of MUCs genes
It has been established that during L. monocytogenes infection signalling events are elicited by virulence factors interacting with host receptor signal transducing molecules (Ireton et al., 1996; Shen et al., 2000). Overexpression of MUC3, MUC4 and MUC12 mucins upon LLO stimulation reported in the present work, might be a new mechanism by which L. monocytogenes promotes signal transduction and/or influences cell growth in human mucin-secreting intestinal cells. Indeed, recent data highlight that the C-termini of human/rat/mouse MUC3, human MUC4 and MUC12 contain two conserved cysteine-rich, EGF-like domains (Williams et al., 1999a,b). It was interesting to note that MUC12 possesses a cytoplasmic tail containing an amino acid sequence, which is similar to motifs recognized by SH2 domain-containing proteins (Songyang et al., 1994). Moreover, the first EGF-like domain in MUC12 shows homology to a number of EGF receptor-binding growth factors. Finally, the rat MUC4 isoform containing the EGF-like domains binds the c-erB-2 growth factor receptor and promotes signalling (McNeer et al., 1997; Williams et al., 1999a).
The LLO-upregulated genes encoding for membrane-associated MUC3 and MUC12, are localized to the human chromosome band 7q22 (Gum et al., 1997; Williams et al., 1999a, b). A recent report described evidence for a link between inflammatory bowel disease and markers on chromosome 7q22 (Satsangi et al., 1996). Moreover, MUC3 has been proposed as a candidate susceptibility gene for inflammatory bowel disease (Kyo et al., 1999). An increase in L. monocytogenes immunoreactivity has been observed in biopsies of patients with inflammatory bowel disease (Liu et al., 1995). Moreover, an association between listeriolysin O and the induction of severe inflammatory disease in rat has been documented (Warner et al., 1996). The present results add interest to the proposed putative link between L. monocytogenes intestinal infection and inflammatory bowel disease.