Corticocortical innervation subtypes of layer 5 intratelencephalic cells in the murine secondary motor cortex.

Feedback projections from the secondary motor cortex (M2) to the primary motor and sensory cortices are essential for behavior selection and sensory perception. Intratelencephalic (IT) cells in layer 5 (L5) contribute feedback projections to diverse cortical areas. Here we show that L5 IT cells participating in feedback connections to layer 1 (L1) exhibit distinct projection patterns, genetic profiles, and electrophysiological properties relative to other L5 IT cells. An analysis of the MouseLight database found that L5 IT cells preferentially targeting L1 project broadly to more cortical regions, including the perirhinal and auditory cortices, and innervate a larger volume of striatum than the other L5 IT cells. We found experimentally that in upper L5 (L5a), ER81 (ETV1) was found more often in L1-preferring IT cells, and in IT cells projecting to perirhinal/auditory regions than those projecting to primary motor or somatosensory regions. The perirhinal region-projecting L5a IT cells were synaptically connected to each other and displayed lower input resistance than contra-M2 projecting IT cells including L1-preferring and nonpreferring cells. Our findings suggest that M2-L5a IT L1-preferring cells exhibit stronger ER81 expression and broader cortical/striatal projection fields than do cells that do not preferentially target L1.

Differential Striatal Axonal Arborizations of the Intratelencephalic and Pyramidal-Tract Neurons: Analysis of the Data in the MouseLight Database.

There exist two major types of striatum-targeting neocortical neurons, specifically, intratelencephalic (IT) neurons and pyramidal-tract (PT) neurons. Regarding their striatal projections, it was once suggested that IT axons are extended whereas PT axons are primarily focal. However, subsequent study with an increased number of well-stained extended axons concluded that such an apparent distinction was spurious due to limited sample size. Recent work using genetically labeled neurons reintroduced the differential spatial extent of the striatal projections of IT and PT neurons through population-level analyses, complemented by observations of single axons. However, quantitative IT vs. PT comparison of a large number of axons remained to be conducted. We analyzed the data of axonal end-points of 161 IT neurons and 33 PT neurons in the MouseLight database (http://ml-neuronbrowser.janelia.org/). The number of axonal end-points in the ipsilateral striatum exhibits roughly monotonically decreasing distributions in both neuron types. Excluding neurons with no ipsilateral end-point, the distributions of the logarithm of the number of ipsilateral end-points are considerably overlapped between IT and PT neurons, although the proportion of neurons having more than 50 ipsilateral end-points is somewhat larger in IT neurons than in PT neurons. Looking at more details, among IT subpopulations in the secondary motor area (MOs), layer 5 neurons and bilateral striatum-targeting layer 2/3 neurons, but not contralateral striatum-non-targeting layer 2/3 neurons, have a larger number of ipsilateral end-points than MOs PT neurons. We also found that IT ipsilateral striatal axonal end-points are on average more widely distributed than PT end-points, especially in the medial-lateral direction. These results indicate that IT and PT striatal axons differ in the frequencies and spatial extent of end-points while there are wide varieties within each neuron type.

Ipsi- and contralateral corticocortical projection-dependent subcircuits in layer 2 of the rat frontal cortex.

In the neocortex, both layer 2/3 and layer 5 contain corticocortical pyramidal cells projecting to other cortices. We previously found that among L5 pyramidal cells of the secondary motor cortex (M2), not only intratelencephalic projection cells but also pyramidal tract cells innervate ipsilateral cortices and that the two subtypes are different in corticocortical projection diversity and axonal laminar distributions. Layer 2/3 houses intratelencephalically projecting pyramidal cells that also innervate multiple ipsilateral and contralateral cortices. However, it remained unclear whether layer 2/3 pyramidal cells can be divided into projection subtypes each with distinct innervation to specific targets. In the present study we show that layer 2 pyramidal cells are organized into subcircuits on the basis of corticocortical projection targets. Layer 2 corticocortical cells of the same projection subtype were monosynaptically connected. Between the contralaterally and ipsilaterally projecting corticocortical cells, the monosynaptic connection was more common from the former to the latter. We also found that ipsilaterally and contralaterally projecting corticocortical cell subtypes differed in their morphological and physiological characteristics. Our results suggest that layer 2 transfers separate outputs from M2 to individual cortices and that its subcircuits are hierarchically organized to form the discrete corticocortical outputs.NEW & NOTEWORTHY Pyramidal cell subtypes and their dependent subcircuits are well characterized in cortical layer 5, but much less is understood for layer 2/3. We demonstrate that in layer 2 of the rat secondary motor cortex, ipsilaterally and contralaterally projecting corticocortical cells are largely segregated. These layer 2 cell subtypes differ in dendrite morphological and intrinsic electrophysiological properties, and form subtype-dependent connections. Our results suggest that layer 2 pyramidal cells form distinct subcircuits to provide discrete corticocortical outputs.

A complex between phosphatidylinositol 4-kinase IIα and integrin α3ß1 is required for N-glycan sialylation in cancer cells.

Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3ß1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3ß1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3ß1.

Distinct effects of ß1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells.

An aberrant expression of integrin ß1 has been implicated in breast cancer progression. Here, we compared the cell behaviors of wild-type (WT), ß1 gene deleted (KO), and ß1 gene restored (Res) MDA-MB-231 cells. Surprisingly, the expression of ß1 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 ß1 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 ß1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light on cancer therapies.

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels.

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits α2,3-sialylation but not α2,6-sialylation. The migratory ability of cells without or with a low level of α2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly α2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of α2,6-sialylation by overexpressing ß-galactoside α2,6-sialyltranferase 1 in the α2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of α2,6-sialylation by knock-out of ß-galactoside α2,6-sialyltranferase 1 in α2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of α2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.

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

Integrin α5ß1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5ß1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5ß1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5ß1 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.

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; Fut8(-/-)) 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 Fut8 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.

ß-Galactoside α2,6-sialyltranferase 1 promotes transforming growth factor-ß-mediated epithelial-mesenchymal transition.

ß-Galactoside α2,6-sialyltranferase 1 (ST6GAL1) catalyzes the addition of terminal α2,6-sialylation to N-glycans. Increased expression of ST6GAL1 has been reported in diverse carcinomas and highly correlates with tumor progression. Here, we report that St6gal1 transcription and α2,6-sialylated N-glycans are up-regulated during TGF-ß-induced epithelial-mesenchymal transition (EMT) in GE11 cells, requiring the Sp1 element within the St6gal1 promoter. Knockdown of St6gal1 strongly suppressed TGF-ß-induced EMT with a concomitant increase in E-cadherin expression, a major determinant of epithelial cell adherens junctions. Conversely, overexpression of ST6GAL1 increased the turnover of cell surface E-cadherin and promoted TGF-ß-induced EMT. Overexpressing ß-galactoside α2,3-sialyltranferase 4 had little influence on EMT, indicating specificity for α2,6-sialylation. The basal mesenchymal phenotype of MDA-MB-231 human breast cancer cells was partially reversed by ST6GAL1 silencing. Moreover, ST6GAL1 knockdown inhibited the phosphorylation of Akt, but not Smad2, suggesting that ST6GAL1 contributes to EMT through a non-Smad signaling pathway. Taken together, our data indicate that ST6GAL1 promotes TGF-ß-dependent EMT as well as maintenance of the mesenchymal state by growth signaling, providing a plausible mechanism whereby up-regulated ST6GAL1 may promote malignant progression.

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

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.