An Intracellular Activation of Smoothened that is Independent of Hedgehog Stimulation in \u3cem\u3eDrosophila\u3c/em\u3e

Smoothened (Smo), a GPCR family protein, plays a critical role in the reception and transduction of Hedgehog (Hh) signal. Smo is phosphorylated and activated on the cell surface; however, it is unknown whether Smo can be intracellularly activated. Here, we demonstrate that inactivation of the ESCRT-III causes dramatic accumulation of Smo in the ESCRT-III/MVB compartment, and subsequent activation of Hh signaling. In contrast, inactivation of ESCRTs 0–II induces mild Smo accumulation in the ESCRT-III/MVB compartment. We provide evidence that Kurtz (Krz), the Drosophila β-arrestin2, acts in parallel with the ESCRTs 0-II pathway to sort Smo to the multivesicular bodies and lysosome-mediated degradation. Additionally, upon inactivation of ESCRT-III, all active and inactive forms of Smo are accumulated. Endogenous Smo accumulated upon ESCRT-III inactivation is highly activated, which is induced by phosphorylation but not sumoylation. Taken together, our findings demonstrate a model for intracellular activation of Smo, raising the possibility for tissue overgrowth caused by an excessive amount, rather than mutation of Smo

SUMO Regulates the Activity of Smoothened and Costal-2 in \u3cem\u3eDrosophila\u3c/em\u3e Hedgehog Signaling

In Hedgehog (Hh) signaling, the GPCR-family protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation and ubiquitination, which ultimately change the cell surface accumulation of Smo. However, it is not clear whether Smo is regulated by other post-translational modifications, such as sumoylation. Here, we demonstrate that knockdown of the small ubiquitin-related modifier (SUMO) pathway components Ubc9 (a SUMO-conjugating enzyme E2), PIAS (a SUMO-protein ligase E3), and Smt3 (the SUMO isoform in Drosophila) by RNAi prevents Smo accumulation and alters Smo activity in the wing. We further show that Hh-induced-sumoylation stabilizes Smo, whereas desumoylation by Ulp1 destabilizes Smo in a phosphorylation independent manner. Mechanistically, we discover that excessive Krz, the Drosophila β-arrestin 2, inhibits Smo sumoylation and prevents Smo accumulation through Krz regulatory domain. Krz likely facilitates the interaction between Smo and Ulp1 because knockdown of Krz by RNAi attenuates Smo-Ulp1 interaction. Finally, we provide evidence that Cos2 is also sumoylated, which counteracts its inhibitory role on Smo accumulation in the wing. Taken together, we have uncovered a novel mechanism for Smo activation by sumoylation that is regulated by Hh and Smo interacting proteins

Hrs Promotes Ubiquitination and Mediates Endosomal Trafficking of Smoothened in Drosophila Hedgehog Signaling

In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation

Identification and verification of novel immune-related ferroptosis signature with excellent prognostic predictive and clinical guidance value in hepatocellular carcinoma

Background: Immunity and ferroptosis often play a synergistic role in the progression and treatment of hepatocellular carcinoma (HCC). However, few studies have focused on identifying immune-related ferroptosis gene biomarkers.Methods: We performed weighted gene co-expression network analysis (WGCNA) and random forest to identify prognostic differentially expressed immune-related genes (PR-DE-IRGs) highly related to HCC and characteristic prognostic differentially expressed ferroptosis-related genes (PR-DE-FRGs) respectively to run co-expression analysis for prognostic differentially expressed immune-related ferroptosis characteristic genes (PR-DE-IRFeCGs). Lasso regression finally identified 3 PR-DE-IRFeCGs for us to construct a prognostic predictive model. Differential expression and prognostic analysis based on shared data from multiple sources and experimental means were performed to further verify the 3 modeled genes’ biological value in HCC. We ran various performance testing methods to test the model’s performance and compare it with other similar signatures. Finally, we integrated composite factors to construct a comprehensive quantitative nomogram for accurate prognostic prediction and evaluated its performance.Results: 17 PR-DE-IRFeCGs were identified based on co-expression analysis between the screened 17 PR-DE-FRGs and 34 PR-DE-IRGs. Multi-source sequencing data, QRT-PCR, immunohistochemical staining and testing methods fully confirmed the upregulation and significant prognostic influence of the three PR-DE-IRFeCGs in HCC. The model performed well in the performance tests of multiple methods based on the 5 cohorts. Furthermore, our model outperformed other related models in various performance tests. The immunotherapy and chemotherapy guiding value of our signature and the comprehensive nomogram’s excellent performance have also stood the test.Conclusion: We identified a novel PR-DE-IRFeCGs signature with excellent prognostic prediction and clinical guidance value in HCC

Neurotensin Regulates Proliferation and Stem Cell Function in the Small Intestine in a Nutrient-Dependent Manner

BACKGROUND & AIMS: Intestinal stem cells (ISCs) are sensitive to dietary alterations and nutrient availability. Neurotensin (NT), a gut peptide localized predominantly to the small bowel and released by fat ingestion, stimulates the growth of intestinal mucosa under basal conditions and during periods of nutrient deprivation, suggesting a possible role for NT on ISC function. METHODS: Leucine-rich repeat-containing G-protein coupled receptor 5-Enhanced Green Fluorescent Protein (Lgr5-EGFP) NT wild type (Nt+/+) and Lgr5-EGFP NT knockout (Nt-/-) mice were fed ad libitum or fasted for 48 hours. Small intestine tissue and crypts were examined by gene expression analyses, fluorescence-activated cell sorting, Western blot, immunohistochemistry, and crypt-derived organoid culture. Drosophila expressing NT in midgut enteroendocrine cells were fed a standard diet or low-energy diet and esg-green fluorescent protein+ ISCs were quantified via immunofluorescence. RESULTS: Loss of NT impaired crypt cell proliferation and ISC function in a manner dependent on nutrient status. Under nutrient-rich conditions, NT stimulated extracellular signal-regulated kinases 1 and 2 signaling and the expression of genes that promote cell-cycle progression, leading to crypt cell proliferation. Under conditions of nutrient depletion, NT stimulated WNT/β-catenin signaling and promoted an ISC gene signature, leading to enhanced ISC function. NT was required for the induction of WNT/β-catenin signaling and ISC-specific gene expression during nutrient depletion, and loss of NT reduced crypt cell proliferation and impaired ISC function and Lgr5 expression in the intestine during fasting. Conversely, the expression of NT in midgut enteroendocrine cells of Drosophila prevented loss of ISCs during nutrient depletion. CONCLUSIONS: Collectively, our findings establish an evolutionarily conserved role for NT in ISC maintenance during nutritional stress. GSE182828

Intrinsic correlation between the fraction of liquidlike zones and the beta relaxation in high-entropy metallic glasses

Lacking the structural information of crystalline solids, the origin of the relaxation dynamics of metallic glasses is unclear. Here, we report the evolution of stress relaxation of high-entropy metallic glasses with distinct ß relaxation behavior. The fraction of liquidlike zones, determined at each temperature by the intensity of stress decay, is shown to be directly related to both the aging process and the spectrum of relaxation modes obtained by mechanical spectroscopy. The results shed light on the intrinsic correlation between the static and dynamic mechanical response in high-entropy and conventional metallic glasses, pointing toward a sluggish diffusion high-entropy effect in the liquid dynamics.Postprint (author's final draft

Human Very Small Embryonic-Like Cells Generate Skeletal Structures, In Vivo

Human very small embryonic-like (hVSEL) cells are a resident population of multipotent stem cells in the bone marrow involved in the turnover and regeneration of tissues. The levels of VSEL cells in blood are greatly increased in response to injury, and they have been shown to repair injured tissues. Adult hVSEL cells, SSEA-4+/CD133+/CXCR4+/Lin?/CD45?, express the pluripotency markers (Oct-4 and Nanog) and may be able to differentiate into cells from all 3 germ lineages. hVSEL cells isolated from blood by apheresis following granulocyte?colony-stimulating factor mobilization were fractionated and enriched by elutriation and fluorescence activated cell sorting. Collagen sponge scaffolds containing 2,000?30,000 hVSEL cells were implanted into cranial defects generated in SCID mice. Analysis by microcomputed tomography showed that a cell population containing VSEL cells produced mineralized tissue within the cranial defects compared with controls at 3 months. Histologic studies showed significant bone formation and cellular organization within the defects compared with cellular or scaffold controls alone. Antibodies to human leukocyte antigens demonstrated that the newly generated tissues were of human origin. Moreover, human osteocalcin was identified circulating in the peripheral blood. There was evidence that some level of hVSEL cells migrated away from the defect site, using quantitative real-time polymerase chain reaction to detect for human-specific Alu sequences. This study demonstrates that hVSEL cells are able to generate human bone tissue in a mouse model of skeletal repair. These studies lay the foundation for future cell-based regenerative therapies for osseous and connective tissue disorders, including trauma and degenerative conditions, such as osteoporosis, fracture repair, and neoplastic repair.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140198/1/scd.2012.0327.pd

Human and Murine Very Small Embryonic-Like Cells Represent Multipotent Tissue Progenitors, In Vitro and In Vivo

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ?60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders. Key Points HuVSEL and MuVSEL cells are capable of differentiating into multiple germline derivatives in vitro and in vivo. MuVSEL cells have limited capacity for self-renewal and neither HuVSEL nor MuVSEL cells formed tumors in immunodeficient animals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140202/1/scd.2013.0362.pd