Qinglei Hang

An oncogenic protein Golgi phosphoprotein 3 up-regulates cell migration via sialylation

Background: Molecular mechanisms of the effect of the GOLPH3 oncogenic protein on tumorigenesis remain unclear. Results: GOLPH3 specifically up-regulates sialylation of integrin N-glycans, promotes sialylation-dependent cell migration, and affects AKT signaling. Conclusion: GOLPH3 affects cell biological functions through a specific regulation of sialylation. Significance: The sialylation of N-glycans is important for functions of GOLPH3. Recently, the Golgi phosphoprotein 3 (GOLPH3) and its yeast homolog Vps74p have been characterized as essential for the Golgi localization of glycosyltransferase in yeast. GOLPH3 has been identified as a new oncogene that is commonly amplified in human cancers to modulate mammalian target of rapamycin signaling. However, the molecular mechanisms of the carcinogenic signaling pathway remain largely unclear. To investigate whether the expression of GOLPH3 was involved in the glycosylation processes in mammalian cells, and whether it affected cell behavior, we performed a loss-of-function study. Cell migration was suppressed in GOLPH3 knockdown (KD) cells, and the suppression was restored by a re-introduction of the GOLPH3 gene. HPLC and LC/MS analysis showed that the sialylation of N-glycans was specifically decreased in KD cells. The specific interaction between sialyltransferases and GOLPH3 was important for the sialylation. Furthermore, overexpression of α2,6-sialyltransferase-I rescued cell migration and cellular signaling, both of which were blocked in GOLPH3 knockdown cells. These results are the first direct demonstration of the role of GOLPH3 in N-glycosylation to regulate cell biological functions.

Loss of α1,6-fucosyltransferase inhibits chemical-induced hepatocellular carcinoma and tumorigenesis by down-regulating several cell signaling pathways

Up‐regulation of core fucosylation catalyzed by α1,6‐fucosyltransferase (Fut8) has been observed in hepatocellular carcinoma (HCC). Here, to explore the role of Fut8 expression in hepatocarcinogensis, we established the chemical‐induced HCC models in the male wild‐type (WT; Fut8+/+), hetero (Fut8+/‐), and knockout (KO; mice by use of diethylnitrosamine (DEN) and pentobarbital (PB). In the Fut8+/+ and Fut8+/‐ mice, multiple large and vascularized nodules were induced with an increased expression of Fut8 after DEN and PB treatment. However, the formation of HCC in Fut8‐/‐ mice was suppressed almost completely. This potent inhibitory effect of Fut8 deficiency on tumorigenesis was also confirmed by the abolished tumor formation of Fnt8 KO human hepatoma cell line cells by use of a xenograft tumor model. Furthermore, loss of the Fut8 gene resulted in attenuated responses to epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the HepG2 cell line, which provides the possible mechanisms for the contribution of Fut8 to hepatocarcinogensis. Taken together, our study clearly demonstrated that core fucosylation acts as a critical functional modulator in the liver and implicated Fut8 as a prognostic marker, as well as a novel, therapeutic target for HCC.—Wang, Y., Fukuda, T., Isaji, T., Lu, J., Im, S., Hang, Q., Gu, W., Hou, S., Ohtsubo, K., Gu, J. Loss of α1,6‐fucosyltransferase inhibits chemical induced hepatocellular carcinoma and tumorigenesis by down‐regulating several cell signaling pathways. FASEB J. 29, 3217‐3227 (2015). www.fasebj.org

Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells

An aberrant expression of integrin β1 has been implicated in breast cancer progression. Here, we compared the cell behaviors of wild-type (WT), β1 gene deleted (KO), and β1 gene restored (Res) MDA-MB-231 cells. Surprisingly, the expression of β1 exhibited opposite effects on cell proliferation. These effects were dependent on cell densities, and they showed an up-regulation of cell proliferation when cells were cultured under sparse conditions, and a down-regulation of cell growth under dense conditions. By comparison with WT cells, the phosphorylation levels of ERK in KO cells were consistently suppressed under sparse culture conditions, but consistently up-regulated under dense culture conditions. The phosphorylation levels of EGFR were increased in the KO cells. By contrast, the phosphorylation levels of AKT were decreased in the KO cells. The abilities for both colony and tumor formation were significantly suppressed in the KO cells, suggesting that β1 plays an important role in cell survival signaling for tumorigenesis. These aberrant phenotypes in the KO cells were rescued in the Res cells. Taken together, these results clearly showed the distinct roles of β1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light on cancer therapies.

Loss of α1,6-Fucosyltransferase Decreases Hippocampal Long Term Potentiation IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING

Background: High expression levels of core fucosylated N-glycans in brain tissues remain unexplained. Results: Loss of core fucosylation enhanced AMPA receptor heteromerization and decreased long term potentiation. Conclusion: Core fucosylation is required for hippocampal long term potentiation. Significance: Core fucosylation may be very important for the neuronal synaptic plasticity that is required for learning and memory. Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8−/−) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8−/− mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8−/− mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8−/− mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca2+ influx and CaMKII and then impair LTP.

Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation

Background: The functions of integrin α5 on cell proliferation and the underlying mechanisms remain unclear. Results: Loss of N-glycosylation on α5 increased the phosphorylation and internalization of EGFR and abolished its inhibitory effects on cell proliferation. Conclusion: Integrin α5 regulates EGFR-mediated signaling through N-glycosylation. Significance: N-Glycosylation plays important roles in the cross-talk between integrins and growth factor receptors. Integrin α5β1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5β1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5β1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5β1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3–5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3–5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors.

A key regulator of cell adhesion: Identification and characterization of important N-glycosylation sites on integrin α5 for cell migration

ABSTRACT The N-glycosylation of integrin α5β1 is thought to control many fundamental aspects of cell behavior, including cell adhesion and migration. However, the mechanism of how N-glycans function remains largely obscure. Here, we used a loss-of-function approach. Wild-type (WT) integrin α5 and N-glycosylation mutant S3-5 (sites 3 to 5) integrin α5, which contains fewer N-glycans, were stably reconstituted in α5 knockout cancer cells. We found that the migration ability of S3-5 cells was decreased in comparison with that of the WT. Interestingly, the levels of phosphorylated focal adhesion kinase and actin stress fiber formation were greatly enhanced in the S3-5 mutant. In a mechanistic manner, the internalization of active but not total integrin α5β1 was inhibited in S3-5 cells, which is a process that is related to the enhanced expression of active integrin α5β1 on the cell surface. Importantly, restoration of N-glycosylation on the β-propeller domain of α5 reinstated the cell migration ability, active α5β1 expression, and internalization. Moreover, these N-glycans are critical for α5–syndecan-4 complex formation. These findings indicate that N-glycosylation on the β-propeller domain functions as a molecular switch to control the dynamics of α5β1 on the cell surface that in turn is required for optimum adhesion for cell migration.

Importance of membrane-proximal N-glycosylation on integrin β1 in its activation and complex formation

N‐Glycosylation of integrin α5β1 plays important roles in cell biologic functions; however, the mechanisms that underlie those roles remain poorly understood. Here, we present evidence that themembrane‐proximal N‐glycosylation on integrin β1 could positively regulate cell migration by promoting β1 activation. The S4–6 β1 mutant contains only 3 N‐glycosylation sites, which are essential for α5 and β1 heterodimer formation, and despite only a small difference in expression levels of α5β1 between wild‐type and S4–6 mutant, cell spreading and migration of the S4–6 mutant was significantly decreased compared with that of control. Consistent with these phenotypes, β1 ‐mediated cellular signaling and its activation were clearly suppressed in the S4–6 mutant. Of note, these developments could be rescued by restoration of N‐glycosylation sites in the membrane‐proximal domain. Further study on the regulatory mechanisms suggested that membrane‐proximal N‐glycosylation is critical for intermolecular interactions between integrin β1 and other cell membrane proteins, such as syndecan‐4 and epidermal growth factor receptor. Moreover, α2, 6‐sialylation is required for β1 activation. These data suggest a novel regulatory mechanism where in N‐glycosy lationnear the cell membrane on β1 may serve as a platform that facilitates its complex formation on the cell membrane, thereby affecting integrin‐mediated functions.—Hou, S., Hang, Q., Isaji, T., Lu, J., Fukuda, T., Gu, J. Importance ofmembrane‐proximal N‐glycosylation on integrin β1 in its activation and complex formation. FASEB J. 30, 4120–4131 (2016). www.fasebj.org

N -Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4

N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3–5,10–14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3–5,10–14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR.

Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation and migration as well as tumor formation

Core fucosylation is one of the most important glycosylation events in the progression of liver cancer. For this study, we used an easily handled L-fucose analog, 2-fluoro-L-fucose (2FF), which interferes with the normal synthesis of GDP-fucose, and verified its potential roles in regulating core fucosylation and cell behavior in the HepG2 liver cancer cell line. Results obtained from lectin blot and flow cytometry analysis clearly showed that 2FF treatment dramatically inhibited core fucosylation, which was also confirmed via mass spectrometry analysis. Cell proliferation and integrin-mediated cell migration were significantly suppressed in cells treated with 2FF. We further analyzed cell colony formation in soft agar and tumor xenograft efficacy, and found that both were greatly suppressed in the 2FF-treated cells, compared with the control cells. Moreover, the treatment with 2FF decreased the core fucosylation levels of membrane glycoproteins such as EGF receptor and integrin β1, which in turn suppressed downstream signals that included phospho-EGFR, -AKT, -ERK, and -FAK. These results clearly described the roles of 2FF and the importance of core fucosylation in liver cancer progression, suggesting 2FF shows promise for use in the treatment of hepatoma.