Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many ...physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-β signaling during breast cancer progression remains largely unknown.
In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-β-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-β1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model.
Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-β-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-β receptor complexes might be core fucosylated by FUT8 to facilitate TGF-β binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis.
Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-β signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.
Many animals respond to threats by releasing alarm pheromones (APs) that warn conspecifics. In mice, detection of the AP 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT) is mediated by chemosensory neurons ...residing in the Grueneberg ganglion (GG) of the anterior nasal region. Although the molecular mechanisms underlying activation of GG neurons by SBT and other substances are still unclear, recent studies have reported an involvement of the transmembrane guanylyl cyclase (GC) subtype GC‐G in chemosensory signaling in the GG. Here, we show that SBT directly binds with high affinity to the extracellular domain of GC‐G and elicits an enhanced enzymatic activity of this protein. In line with this finding, heterologous expression of GC‐G renders cells responsive to SBT while activation by SBT was strongly attenuated in GG neurons from GC‐G‐deficient mice. Consistently, SBT‐induced fear‐associated behaviors, SBT‐evoked elevated blood pressure, and increased serum levels of the stress hormone corticosterone were clearly reduced in GC‐G‐knockout animals compared to wild‐type mice. These observations suggest that GC‐G serves as an unusual receptor in GG neurons mediating the detection of the volatile AP substance SBT.
Synopsis
The transmembrane guanylyl cyclase GC‐G serves as a receptor for the alarm pheromone compound 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT) in Grueneberg ganglion neurons of the murine nose.
SBT that is released by mice in stressful situations binds with high affinity to the extracellular domain of GC‐G and elicits an enhanced enzymatic activity of this protein, leading to an increased synthesis of the second messenger substance cyclic guanosine monophosphate (cGMP).
Heterologous expression of GC‐G and its downstream effector protein, the cyclic nucleotide‐gated ion channel CNGA3, in a human cell line renders these cells responsive to SBT.
SBT‐induced activation of Grueneberg ganglion neurons is markedly attenuated in GC‐G‐deficient mice.
Innate fear behaviors, elevated blood pressure as well as increased serum concentrations of the stress hormone corticosterone that are elicited by SBT in wild type mice are significantly reduced in GC‐G‐knockout animals.
Gain‐ and loss‐of‐function studies in mouse cells and knock‐out animals unequivocally define the transmembrane guanylyl cyclase GC‐G as the receptor binding to and mediating the stress‐associated effects of the alarm pheromone SBT.
Background
Cav3.2 is a T‐type calcium channel that causes low‐threshold exocytosis. T‐type calcium channel blockers reduce platelet granule exocytosis and aggregation. However, studies of the T‐type ...calcium channel in platelets are lacking.
Objective
To examine the expression and role of Cav3.2 in platelet function.
Methods
Global Cav3.2−/− and platelet‐specific Cav3.2−/− mice and littermate controls were used for this study. Western blot analysis was used to detect the presence of Cav3.2 and activation of the calcium‐responsive protein extracellular signal‐regulated kinase (ERK). Fura‐2 dye was used to assess platelet calcium. Flow cytometry and light transmission aggregometry were used to evaluate platelet activation markers and aggregation, respectively. FeCl3‐induced thrombosis and a microfluidic flow device were used to assess in vivo and ex vivo thrombosis, respectively.
Results
Cav3.2 was expressed in mouse platelets. As compared with wild‐type controls, Cav3.2−/− mouse platelets showed reduced calcium influx. Similarly, treatment with the T‐type calcium channel inhibitor Ni2+ decreased the calcium influx in wild‐type platelets. As compared with controls, both Cav3.2−/− and Ni2+‐treated wild‐type platelets showed reduced activation of ERK. ATP release, P‐selectin exposure, and αIIbβ3 activation were reduced in Cav3.2−/− and Ni2+‐treated wild‐type platelets, as was platelet aggregation. On in vivo and ex vivo thrombosis assay, Cav3.2 deletion caused delayed thrombus formation. However, tail bleeding assay showed intact hemostasis.
Conclusion
These results suggest that Cav3.2 is required for the optimal activation of platelets.
The biology of SCUBE Lin, Yuh-Charn; Sahoo, Binay K; Gau, Shiang-Shin ...
Journal of biomedical science,
05/2023, Letnik:
30, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The SCUBE Signal peptide-Complement C1r/C1s, Uegf, Bmp1 (CUB)-Epithelial growth factor domain-containing protein family consists of three proteins in vertebrates, SCUBE1, 2 and 3, which are highly ...conserved in zebrafish, mice and humans. Each SCUBE gene encodes a polypeptide of approximately 1000 amino acids that is organized into five modular domains: (1) an N-terminal signal peptide sequence, (2) nine tandem epidermal growth factor (EGF)-like repeats, (3) a large spacer region, (4) three cysteine-rich (CR) motifs, and (5) a CUB domain at the C-terminus. Murine Scube genes are expressed individually or in combination during the development of various tissues, including those in the central nervous system and the axial skeleton. The cDNAs of human SCUBE orthologs were originally cloned from vascular endothelial cells, but SCUBE expression has also been found in platelets, mammary ductal epithelium and osteoblasts. Both soluble and membrane-associated SCUBEs have been shown to play important roles in physiology and pathology. For instance, upregulation of SCUBEs has been reported in acute myeloid leukemia, breast cancer and lung cancer. In addition, soluble SCUBE1 is released from activated platelets and can be used as a clinical biomarker for acute coronary syndrome and ischemic stroke. Soluble SCUBE2 enhances distal signaling by facilitating the secretion of dual-lipidated hedgehog from nearby ligand-producing cells in a paracrine manner. Interestingly, the spacer regions and CR motifs can increase or enable SCUBE binding to cell surfaces via electrostatic or glycan-lectin interactions. As such, membrane-associated SCUBEs can function as coreceptors that enhance the signaling activity of various serine/threonine kinase or tyrosine kinase receptors. For example, membrane-associated SCUBE3 functions as a coreceptor that promotes signaling in bone morphogenesis. In humans, SCUBE3 mutations are linked to abnormalities in growth and differentiation of both bones and teeth. In addition to studies on human SCUBE function, experimental results from genetically modified mouse models have yielded important insights in the field of systems biology. In this review, we highlight novel molecular discoveries and critical directions for future research on SCUBE proteins in the context of cancer, skeletal disease and cardiovascular disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE—SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), expressed on the endothelial cell surface, functions as a novel coreceptor for VEGFR2 (vascular endothelial growth factor ...receptor 2) and enhances VEGF-induced signaling in adult angiogenesis. However, whether SCUBE2 plays a role in pathological angiogenesis and whether anti-SCUBE2 antibody is an effective strategy for blocking tumor angiogenesis remain unknown. The aim of this study was to investigate the pathological role and targeting therapy of SCUBE2 in tumor vasculature.
APPROACH AND RESULTS—Immunohistochemistry revealed that SCUBE2 is highly expressed in endothelial cells of numerous carcinomas. Genetic endothelial cell knockout of SCUBE2 and pharmacological inhibition with the anti-SCUBE2 monoclonal antibody SP.B1 significantly reduced xenograft tumor growth, decreased tumor vascular density, increased apoptosis, and decreased the proliferation of tumor cells. Mechanistic studies revealed that SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing its cell surface level and inhibiting the binding of and downstream signaling of VEGF, including VEGFR2 phosphorylation and AKT/MAPK (mitogen-activated protein kinase) activation. Importantly, dual combination therapy with anti-SCUBE2 monoclonal antibody and anti-VEGF antibody or chemotherapy was more effective than single-agent therapy.
CONCLUSIONS—Endothelial cell surface SCUBE2 is a VEGFR2 coreceptor essential for pathological tumor angiogenesis, and anti-SCUBE2 monoclonal antibody acting as an internalization inducer may provide a potent combination therapy for tumor angiogenesis.
Transmembrane guanylyl cyclases (GCs), with activity regulated by peptide ligands and/or calcium‐binding proteins, are essential for various physiological and sensory processes. The mode of ...activation of the GC subtype GC‐G, which is expressed in neurons of the Grueneberg ganglion that respond to cool temperatures, has been elusive. In searching for appropriate stimuli to activate GC‐G, we found that its enzymatic activity is directly stimulated by cool temperatures. In this context, it was observed that dimerization/oligomerization of GC‐G, a process generally considered as critical for enzymatic activity of GCs, is strongly enhanced by coolness. Moreover, heterologous expression of GC‐G in cultured cells rendered these cells responsive to coolness; thus, the protein might be a sensor for cool temperatures. This concept is supported by the observation of substantially reduced coolness‐induced response of Grueneberg ganglion neurons and coolness‐evoked ultrasonic vocalization in GC‐G‐deficient mouse pups. GC‐G may be a novel thermosensory protein with functional implications for the Grueneberg ganglion, a sensory organ responding to cool temperatures.
Synopsis
Many membrane proteins respond to extracellular ligands, but sensory nerves use specialized sensors also to report environmental temperatures. The transmembrane receptor guanylyl cyclase subtype GC‐G, expressed in Grueneberg ganglion neurons of the murine nose, is a novel type of molecular sensor for cool ambient temperatures in mammals.
GC‐G expression in mouse is confined to Grueneberg ganglion neurons of the murine nose.
Cool temperature stimulates the enzymatic activity of the GC‐G guanylyl cyclase by inducing its dimerization/oligomerization.
Coolness‐induced stimulation of the thermosensory GC‐G guanylyl cyclase activates the Grueneberg ganglion neurons by increasing the cGMP concentration and opening cGMP‐dependent Ca2+‐permeable channels for elevating intracellular Ca2+ levels.
Independent of more classical sensors like the coolness‐activated TRPM8 ion channel, GC‐G is critical for coolness‐evoked ultrasound‐vocalization in abandoned mouse pups to elicit maternal‐care behaviors.
The membrane‐spanning receptor guanylyl cyclase subtype GC‐G, specifically expressed in the Grueneberg ganglion neurons in the nose, is a novel thermosensor for cool ambient temperatures in mammals in vivo.
Hair follicle stem cells are regulated by dermal papilla fibroblasts, their principal signaling niche. Overactivation of Hedgehog signaling in the niche dramatically accelerates hair growth and ...induces follicle multiplication in mice. On single-cell RNA sequencing, dermal papilla fibroblasts increase heterogeneity to include new Wnt5a
states. Transcriptionally, mutant fibroblasts activate regulatory networks for Gli1, Alx3, Ebf1, Hoxc8, Sox18, and Zfp239. These networks jointly upregulate secreted factors for multiple hair morphogenesis and hair-growth-related pathways. Among these is non-conventional TGF-β ligand Scube3. We show that in normal mouse skin, Scube3 is expressed only in dermal papillae of growing, but not in resting follicles. SCUBE3 protein microinjection is sufficient to induce new hair growth, and pharmacological TGF-β inhibition rescues mutant hair hyper-activation phenotype. Moreover, dermal-papilla-enriched expression of SCUBE3 and its growth-activating effect are partially conserved in human scalp hair follicles. Thus, Hedgehog regulates mesenchymal niche function in the hair follicle via SCUBE3/TGF-β mechanism.
Activation of metastatic reprogramming is critical for tumour metastasis. However, more detailed knowledge of the underlying mechanism is needed to enable targeted intervention. Here, we show that ...paraspeckle component 1 (PSPC1), identified in an aberrant 13q12.11 locus, is upregulated and associated with poor survival in patients with cancer. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis in multiple cell types and in spontaneous mouse cancer models. PSPC1 is the master activator for transcription factors of EMT and stemness and accompanies c-Myc activation to facilitate tumour growth. PSPC1 increases transforming growth factor-β1 (TGF-β1) secretion through an interaction with phosphorylated and nuclear Smad2/3 to potentiate TGF-β1 autocrine signalling. Moreover, PSPC1 acts as a contextual determinant of the TGF-β1 pro-metastatic switch to alter Smad2/3 binding preference from tumour-suppressor to pro-metastatic genes. Having validated the PSPC1-Smads-TGF-β1 axis in various cancers, we conclude that PSPC1 is a master activator of pro-metastatic switches and a potential target for anti-metastasis drugs.
We recently showed that fucosyltransferase 8 (FUT8)-mediated core fucosylation of transforming growth factor-β receptor enhances its signaling and promotes breast cancer invasion and metastasis. ...However, the complete FUT8 target glycoproteins and their downstream signaling networks critical for breast cancer progression remain largely unknown.
We performed quantitative glycoproteomics with two highly invasive breast cancer cell lines to unravel a comprehensive list of core-fucosylated glycoproteins by comparison to parental wild-type and FUT8-knockout counterpart cells. In addition, ingenuity pathway analysis (IPA) was performed to highlight the most enriched biological functions and signaling pathways mediated by FUT8 targets. Novel FUT8 target glycoproteins with biological interest were functionally studied and validated by using LCA (Lens culinaris agglutinin) blotting and LC-MS/MS (liquid chromatography-tandem mass spectrometry) analysis.
Loss-of-function studies demonstrated that FUT8 knockout suppressed the invasiveness of highly aggressive breast carcinoma cells. Quantitative glycoproteomics identified 140 common target glycoproteins. Ingenuity pathway analysis (IPA) of these target proteins gave a global and novel perspective on signaling networks essential for breast cancer cell migration and invasion. In addition, we showed that core fucosylation of integrin αvβ5 or IL6ST might be crucial for breast cancer cell adhesion to vitronectin or enhanced cellular signaling to interleukin 6 and oncostatin M, two cytokines implicated in the breast cancer epithelial-mesenchymal transition and metastasis.
Our report reveals a comprehensive list of core-fucosylated target proteins and provides novel insights into signaling networks crucial for breast cancer progression. These findings will assist in deciphering the complex molecular mechanisms and developing diagnostic or therapeutic approaches targeting these signaling pathways in breast cancer metastasis.
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