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Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium‐derived peptide (PEDF) short peptide 44‐mer could effectively expand LSCs and maintain them in a stem‐cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44‐mer. In mice subjected to focal limbal injury, 44‐mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44‐mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44‐mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44‐mer‐induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44‐mer‐induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh‐Gli pathway driven by ATGL/STAT3 signalling accounts for the 44‐mer‐mediated LSC proliferation.  相似文献   

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Patching the gaps in Hedgehog signalling   总被引:2,自引:0,他引:2  
The Hedgehog (Hh) pathway plays central roles in animal development and stem-cell function. Defects in Hh signalling lead to birth defects and cancer in humans. The first and often genetically damaged step in this pathway is the interaction between two membrane proteins - Patched (Ptc), encoded by a tumour suppressor gene, and Smoothened (Smo), encoded by a proto-oncogene. Recent work linking Hh signalling to sterol metabolites and protein-trafficking events at the primary cilium promises to shed light on the biochemical basis of how Patched inhibits Smoothened, and to provide new avenues for cancer treatment.  相似文献   

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The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gαi proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gαi protein activity appears to significantly reduce Hh pathway activity in Ptc−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gαi protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gαi protein, Gαi2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Gαi activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.  相似文献   

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The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.  相似文献   

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Hedgehog (Hh) signalling plays a crucial role in the development and patterning of many tissues in both vertebrates and invertebrates. Aberrations in this pathway lead to severe developmental defects and cancer. Hh signal transduction in receiving cells is a well studied phenomenon; however questions still remain concerning the mechanism of repression of the pathway activator Smoothened (Smo) in the absence of Hh. Here we describe a novel repressor of the Hh pathway, Target of Wingless (Tow). Tow represents the Drosophila homolog of a conserved uncharacterised protein family. We show that Tow acts in Hh receiving cells, where its overexpression represses all levels of Hh signalling, and that this repression occurs upstream or at the level of Smo and downstream of the Hh receptor Patched (Ptc). In addition, we find that like Ptc, overexpression of Tow causes an accumulation of lipophorin in the wing disc. We demonstrate that loss of tow enhances different ptc alleles in a similar manner to another pathway repressor, Suppressor of Fused (SuFu), possibly through mediating Ptc dependant lipophorin internalisation. Combined, these results demonstrate that Tow is an important novel regulator of the Hh pathway in the wing imaginal disc, and may shed light on the mechanism of Ptc repression of Smo.  相似文献   

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A series of aminothiazole derivatives bearing the benzimidazole moiety were synthesized and evaluated in Gli luciferase reporter assays. Lead optimization led to the discovery of potent hedgehog pathway antagonist 18 (2‐[3‐(1H‐benzimidazol‐2‐yl)‐4‐chloroanilino]‐N‐[4‐(trifluoromethyl)phenyl]‐1,3‐thiazole‐4‐carboxamide), with IC50 values in nanomolar range. The molecular basis ascribed to hindering sonic hedgehog‐driven Smoothened (Smo) localization within the primary cilium (PC). Moreover, compound 18 inhibited Gli1 mRNA expression in mutant Smo cell line and displayed moderate cytotoxicity against DAOY cancer cell.  相似文献   

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Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo's actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.  相似文献   

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