The present study described the impact of EGC upon the transcriptome of proliferating Caco-2 cells in a validated non-contact co-culture model of EGC and IEC [12
]. The results obtained confirmed the known role of EGC in the control of some IEB functions and, more interestingly, extended their role in the control of novel major IEB and IEC functions. This study further reinforced the emerging concept that EGC are an important component of the IEB environment with major protective effects. Indeed, the major pathways regulated by EGC in IEC identified with microarrays lead to enhanced cell adhesion, differentiation, and motility, which could favor repair, and reduced cell proliferation.
An important result of this study is the putative identification of genes involved in the anti-proliferative effects of EGC. Indeed, EGC have been shown to have potent anti-proliferative effects upon IEC [11
]. Interestingly, these effects were associated with an induction of a cell cycle blockade in the G0/G1 phase [11
] but were not associated with significant cell death [12
]. These results are globally confirmed, as there was no clear trend in the EGC-induced modulation of genes controlling cell survival in IEC but a trend toward an up-regulation of the expression of genes involved in anti-proliferative pathways.
A major finding of our study is that EGC regulated the expression of genes involved in cell adhesion and differentiation toward a global increase of IEC adhesive properties. These results can be analyzed in view of the known effects of EGC upon IEB. Indeed, in vitro
studies have shown that EGC increase IEB resistance and decrease IEB paracellular permeability [13
]. In the present study, we also demonstrated that EGC could increase global IEB adhesion, in part by increasing cell-to-matrix adhesion. These results are in agreement with in vivo
data showing that selective lesions of EGC lead to increased paracellular permeability and major IEB breakdown associated with the development of intestinal inflammation. However, the role of the molecular actors involved in these processes such as fibronectin, laminin or cytokeratin remains to be investigated. EGC might also favor barrier integrity by increasing its resistance to inflammatory stress either by its ability to down-regulate inflammatory genes such as CARD12
or by increasing IEC production of anti-inflammatory mediators such as VIP [82
Another important finding of this study is the observation that EGC might regulate IEC metabolism. In particular, EGC up-regulated genes involved in lipid metabolism such as AADAC
, encoding respectively the arylacetamide deacetylase, monoglyceride lipase (MGL) and Apolipoprotein H [84
]. Interestingly, inhibitors of MGL which is a serine hydrolase that converts 2-arachidonoylglycerol, a ligand of canabinoid receptors, to fatty acids and glycerol, increased gut transit time [87
] but its impact on IEB functions remain unknown. EGC also modulated the expression of genes involved in protein metabolism such as CTSH
that encodes cathepsin H, a lysosomal cysteine proteinase [88
]. In addition, EGC increased the expression of genes involved in arginine metabolic pathway that are SLC7A7
, which encode respectively for the cationic amino acid transporter y(+)LAT1 and the argininosuccinate synthetase, enzyme catalyzing the penultimate step of the arginine biosynthetic pathways. The functional impact of EGC upon IEC metabolism needs to be investigated in future studies.
Regulation of IEB functions by EGC occurs mainly via
paracrine pathways. The majority of EGC effects upon IEB functions are reproduced by glial-derived conditioned medium. In addition, various mediators have been identified as being involved in the control of cell proliferation or paracellular permeability. Our study supports the role of mediators such as TGF-β1 as a regulator of gene pathways modulated by EGC in IEC. In fact, TGF-β1 has been shown to increase the expression of FAK [43
], TGFBI [89
] or VIP [90
]. EGC have also been shown to produce IL-6 [91
]. IL-6 has recently been identified as a key molecule involved in IEB barrier protection via
increasing both cytokeratin 8 and cytokeratin 18 proteins expression [92
], whose mRNA expression were induced by EGC in IEC in our study. In this context, knowledge of genes modulated by EGC could direct future efforts aimed at identifying novel glial mediators involved in EGC control of IEB functions. Our data also further suggest that EGC differentially regulate some IEB functions as compared to fibroblasts, although comparison has only been performed on a limited set of genes and one cannot fully rule out that species differences could also be involved (fibroblasts of human origin vs.
enteric glia of rat origin). However, these differences are consistent with the observation that while EGC have anti-proliferative effects on both human and rat IEC [12
], fibroblasts increase IEC proliferation [93
Collectively, our data support the concept that EGC play a key protective role upon IEB homeostasis by reinforcing global barrier functions. Additionally, our study reinforces data suggesting that enteric glia lesions and/or functional defects could be involved in the development of pathologies with altered barrier (such as inflammatory bowel diseases or colorectal cancer) and also be associated with increased barrier susceptibility to pathogen aggression.