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  • Intestinal epithelium renewal is tightly controlled by Hedge


    Intestinal epithelium renewal is tightly controlled by Hedgehog genes. Three Hedgehog genes are highly conserved in mouse and human, including Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) [13]. Hedgehogs bind to Patched (PTCH) [[14], [15], [16]], which unlike conventional receptors, does not transmit the Hedgehog signal to the intracellular components but alleviates the inhibitory effect of PTCH on another membrane receptor, the seven-transmembrane protein Smoothened (SMO) [[17], [18], [19]]. In the absence of ligand (Ihh, Shh, or Dhh), Hh-ligand–receptor–PTCH suppresses SMO, and thus inhibits the cleavage of Gli [20,21]. When ligands bind PTCH, SMO suppression is relieved, and Gli is cleaved and transported to the nucleus resulting in the transcription of Hh TNF-alpha, recombinant human protein target genes including PTCH and Gli themselves and cell proliferation-related genes including CyclinD1 [21,22]. PTCH1, PTCH2, Gli1, and Hedgehog-interacting protein can be used as readouts of pathway activity [23,24]. Ihh is produced by differentiated TNF-alpha, recombinant human protein in the adult colon [25] and small intestine [26]. Evidence indicates that it acts as a negative feedback signal for proliferating cells in the crypt [27]. Thus, Hedgehog signaling might negatively regulate intestinal epithelium proliferation and reduce the transit amplifying region of the crypt in adult mice. However, the role of Ihh-mediated proliferation in CFTR-mutant intestines is unknown. Wnt/β-catenin signaling is implicated in the control of stem cell activity, cell proliferation, and cell survival in the gastrointestinal epithelium. Interestingly, β-catenin has been shown to regulate the Ihh pathway in colon cancer [28,29]. We found that CFTR interacts with β-catenin and stabilizes it in epithelial cells and embryonic stem cells [30,31]. Given the reported involvement of Ihh in regulating intestinal proliferation, we hypothesized that CFTR might regulate Ihh activity through the β-catenin pathway, dysfunction of which might lead to inhibition of the hedgehog pathway and exaggerated proliferation, which is observed in CFTR-mutant intestines. We tested this hypothesis and focused on potential correlations between CFTR and the Ihh pathway.
    Materials and methods
    Discussion Our results, for the first time, reveal that CFTR regulates Ihh through β-catenin in the mouse intestine and human intestinal epithelial cells. CFTR dysfunction increases the degradation of β-catenin and inhibits the hedgehog pathway, leading to TCF4 and CyclinD1 transcriptional activation, thereby promoting proliferation of the intestinal epithelium (Fig. 8). Here, we investigated the roles of CFTR in mouse intestinal proliferation. We found that crypt depth was increased significantly in the ΔF508 mouse small intestine, which was accompanied by the marked upregulation of PCNA, indicating activation of proliferation. We further demonstrated that proliferation in the ΔF508 mouse small intestine and CFTR-knockdown Caco2 cells was caused by inhibition of hedgehog signaling due to β-catenin degradation, with disrupted CFTR–β-catenin interactions. The hedgehog pathway, which regulates the proliferation of crypts in the intestine, was markedly downregulated in ΔF508 mouse small intestines and CFTR-knockdown Caco2 cells. We also demonstrated that β-catenin could regulate the hedgehog pathway. Activation of β-catenin in vitro and in vivo reversed the downregulation of Gli1, which is a key transcription factor of the hedgehog pathway. Importantly, activating β-catenin in vivo using LiCl reduced the numbers of proliferating cells in the intestinal crypts of ΔF508 mice. Recent research has clearly demonstrated that the role of Hedgehog signaling is not restricted to intestinal development, and that it is important for the maintenance of adult small intestine homeostasis [39]. Ihh is produced by the superficial epithelium and signals to the underlying mesenchyme where it targets smooth muscle cells, myofibroblast-like cells, and possibly myeloid cells [19]. Ihh seems to be a key signal emitted by the superficial epithelium to indicate its integrity [40]. Loss of this signal itself is sufficient to trigger not only the activation of an epithelial repair program and the influx of fibroblasts and macrophages, but also mesenchymal immune response activation [26]. It has been reported that undifferentiated Caco2 cells express lower levels of CFTR mRNA, whereas a 10-fold increase was observed in differentiated cells [41]. Our data also showed that CFTR was increased with the increase in Ihh after differentiation or became polarized, and that Ihh was downregulated in CFTR-knockdown Caco2 cells (Supplementary Fig. S1). These results suggest that Ihh can be regulated by CFTR during differentiation.