In a1,3-FucT-VII-transfected cells, expression of insulin receptor InR a- and b subunits and epidermal growth factor receptor EGFR on the cell surface and in cells, as well as the sialyl
Trang 1molecules of the insulin receptor pathway
Qiu-yan Wang1, Ying Zhang1, Hai-jiao Chen2, Zong-hou Shen1and Hui-li Chen1
1 Key Laboratory of Glycoconjugate Research, Shanghai Medical College, Fudan University, Shanghai, China
2 Department of Urology, Zhong-shan Hospital, Fudan University, Shanghai, China
Glycosylation is important and the most common form
of post-translational modification that regulates many
aspects of protein function [1,2] In recent years,
increased attention has been paid to the relationship
between structural changes in surface glycans and sur-face receptor signaling It has been reported [3] that overexpression of N-acetylglucosaminyltransferase (GnT)-III introducing a bisecting N-acetylglucosamine
Keywords
epidermal growth factor receptor;
a1,3-fucosyltransferase-VII; human
hepatocarcinoma cell line; insulin receptor;
signaling molecules
Correspondence
H Chen, Key Laboratory of Glycoconjugate
Research, Ministry of Health, Department of
Biochemistry, Shanghai Medical College,
Fudan University, Shanghai 200032, China
Fax: + 86 21 6416 4489
Tel: + 86 21 5423 7223
E-mail: hlchen@shmu.edu.cn
(Received 19 July 2006, revised 13 November
2006, accepted 17 November 2006)
doi:10.1111/j.1742-4658.2006.05599.x
Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of a1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively In a1,3-FucT-VII-transfected cells, expression of insulin receptor (InR) a- and b subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLex, the product of a1,3-FucT-VII) content of the EGFR were unchanged However the level of SLex on the InR a subunit (InR-a) was increased dramatically Tyrosine autophosphory-lation of InR-b , but not EGFR, was elevated Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser⁄ Thr phos-phorylation of protein kinase B (PKB; Akt), p42⁄ 44 mitogen-activated pro-tein kinase (MAPK), MAPK kinase (MEK), and the propro-tein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and b-catenin were also upregulated The activities of PKB and transcription factor TCF were concomitantly sti-mulated Upregulation of InR signaling molecules and their phosphoryla-tion was correlated with the level of SLex on InR-a and a1,3-FucT-VII expression in cells In addition, the phosphorylation intensity and differ-ence in phosphorylation intensity between cells with different levels of a1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLex Furthermore, insulin-induced signaling was facilitated in a1,3-FucT-VII-transfected cells, particularly FucTVII-H These findings provide strong evidence that a1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling path-way These effects are likely mediated by its product, SLex, on the glycans
of the InR This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling
Abbreviations
CDK, cyclin-dependent kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FucT, fucosyltransferase; GlcNAc, N-acetylglucosamine; GnT, N-acetylglucosaminyltransferase; InR, insulin receptor; IRS-1, insulin receptor substrate-1; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; NGF, nerve growth factor; PDK-1, phosphoinositide-dependent kinase-1; PKB, protein kinase B (Akt); PKN, novel protein kinase; TGF, transforming growth factor.
Trang 2(GlcNAc) into the N-glycans of epidermal growth
fac-tor recepfac-tor (EGFR) in U373 MG glioma cells led to
decreased epidermal growth factor (EGF) binding and
autophosphorylation of EGFR, as well as reduced cell
proliferation upon EGF stimulation It has also been
reported [4] that overexpression of GnT-III in
pheo-chromocytoma PC12 cells inhibited transient tyrosine
phosphorylation and dimerization of the nerve growth
factor (NGF) receptor (Trk) upon stimulation with
NGF and resulted in blockage of the neurite outgrowth
during differentiation We previously found [5] that
transfection of the sense cDNA of GnT-V, an enzyme
associated with cancer progression and metastasis, into
human H7721 hepatocarcinoma cells resulted in an
increase in the level of GlcNAcb1,6Mana1,6-branch
(GnT-V product) on the N-glycans of EGFR; this
pro-moted EGF binding and tyrosine autophosphorylation,
but had little effect on expression of the EGFR protein
The phosphorylation (at T308, S473 and tyrosine
resi-dues) and activity of protein kinase B (PKB; Akt), as
well as the phosphorylation of p42⁄ 44
mitogen-activa-ted protein kinase (MAPK; ERK-1⁄ 2) and MAPK
kinase (MEK) before and after EGF stimulation, were
also upregulated Conversely, in H7721 cells expressing
antisense GnT-V (GnTV-AS), the results were the
oppo-site of those seen in GnT-V sense cDNA
(GnTV-S)-transfected cells After GnT-V-(GnTV-S)-transfected H7721 cells
were treated with 1-deoxymannojirimycin, an inhibitor
of N-glycan processing at the high mannose stage, or
antibody against the extracellular glycan domain of
EGFR, the increase in PKB activity and MAPK
phos-phorylation were significantly blocked, and the
differ-ences in PKB activity and MAPK phosphorylation
among GnTV-S, GnTV-AS and mock-transfected cells
(cells transfected with empty vector) were attenuated
significantly These findings indicated that the altered
signaling after GnTV-S or GnTV-AS transfection was
mediated by a structural change in N-glycans on the
EGFR [5] Furthermore, Guo et al [6] reported that
transfection of GnT-V into human fibrosarcoma
HT1080 and mouse NIH 3T3 cells to increase the
Glc-NAcb1,6-branch on N-cadherin inhibited signaling
between N-cadherin and ERK1⁄ 2, and consequently
reduced calcium-dependent cell–cell adhesion mediated
by N-cadherin These results provide evidence that the
N-cadherin signaling pathway is also influenced by the
glycan structures on N-cadherin Wang et al [7]
repor-ted that in embryonic fibroblast cells deprived of
a1,6-fucosyltransferase (FucT-VIII), an enzyme responsible
for the synthesis of core fucose on N-glycans,
EGF-induced phosphorylation of EGFR and
EGFR-mediated JNK or ERK activation were suppressed
Taniguchi [8] also discovered that signal transduction
of the transforming growth factor b1 (TGF b1) recep-tor was deficient in FucT-VIII knockout mice, leading
to emphysema-like phenotypes in the lung These results show that the core fucose on N-glycans is essen-tial for EGF and TGF b1 signaling However, all the above-mentioned structural changes in receptor glycans are located in the core portion of N-glycan, and whe-ther alteration of the terminal residue on the outer chain of either N- or O-glycan can also modify surface receptor signaling remains unclear
It has been documented that sialyl Lewis antigens (SLe) expressed on the surface of cancer cells, such as SLex [SAa2,3 Galb1,4 (Fuca1,3) GlcNAc-] and SLea [SAa2,3 Galb1,3 (Fuca1,4) GlcNAc-], are another kind
of glycan structure involved in metastasis, and which can serve as the ligands for E- or P-selectin expressed
on the surface of vascular endothelial cells and mediate the adhesion of malignant cells to vascular endothe-lium [9–11] The final fucosylation step in Lewis anti-gen synthesis is catalyzed by a1,3-fucosyltransferase (a1,3-FucT) To date, six a1,3-FucTs (III to VII and IX) have been identified Each enzyme has a unique acceptor–substrate binding pattern, and each generates
a unique range of fucosylated products [12,13] Among these, a1,3-FucT-VII, which is expressed mainly in leu-kocytes, catalyzes sialylated substrate and produces SLexas its only product [14] SLexis always located at the terminus of sugar chains, and a1,3-FucT-VII may
be considered a terminal glycosyltransferase that cata-lyzes the final step in sugar-chain processing
In our laboratory, it has been found that the surface SLex and cellular a1,3-FucT-VII of H7721 cells is up- and downregulated by transfection of the metasta-sis-promoting gene c-erbB2⁄ neu and the metastasis-suppressive gene nm23-H1, respectively [15–17] In addition, surface SLex was increased when H7721 cells were treated with proliferative inducers, and decreased after treatment with differentiative inducers [18] The change in SLex level was proportional to a1,3-FucT-VII expression Moreover, the ex vivo metastatic potential was positively correlated with surface SLex and cellular a1,3-FucT-VII levels, and could be inhib-ited by a mAb (KM93) against surface SLex [15,18] Further studies have shown that insulin also enhanced expression of SLex and a1,3-FucT-VII and the meta-static potential of H7721 cells [19] In addition, our group recently found that expression of cyclin-depend-ent kinase (CDK) inhibitor, p27Kip1 protein, was decreased in H7721 cells transfected with a1,3-FucT-VII cDNA Uninhibited CDK2 resulted from a reduc-tion in the p27Kip1-stimulated phosphorylation of retinoblastoma protein, facilitating G1⁄ S transition and increasing the growth rate in the cells These effects
Trang 3were correlated with an increase in surface SLex on
H7721 cells expressing different a1,3-FucT-VII
intensi-ties, and could be blocked by SLex antibody in a
dose-dependent manner, indicating that p27Kip1 expression
was influenced by a1,3-FucT-VII and its product SLex
[20] Therefore, it is interesting to study whether
trans-fection of a1,3-FucT-VII can also affect the function
of some other surface receptors and subsequently
result in altered receptor signaling
Insulin receptor (InR) was selected to study the
effects of a1,3FucT-VII on its expression, SLexcontent
and tyrosine autophosphorylation InR contains two
extracellular carbohydrate-containing a subunits and
two b subunits with cytoplasmic tyrosine kinase
activ-ity [21] InR results were compared with those from
EGFR, which also contains N-glycans on its
extracel-lular domain and tyrosine autophosphorylation sites
at its intracellular domain [22] Furthermore, insulin
receptor substrate-1 (IRS-1), PKB,
phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase
(PKN), p42⁄ 44 MAPK and MEK were analyzed as
the signaling molecules involved in InR signaling
[19,23] Expression of b catenin and its downstream
transcription factor TCF in the Wnt signaling pathway
[24], which cross-talks with the InR pathway was
also studied Mock cells transfected with the vector pcDNA3.1 were used as controls
Results
Characterization of two a1,3-FucT-VII-transfected cell lines
As shown in Fig 1A,B, a1,3-FucT-VII mRNA was increased significantly in H7721 cells transfected with a1,3-FucT-VII cDNA In FucTVII-M (moderate expression) and FucTVII-H (high expression) cells, it was upregulated to 373.3 and 613.3% of the mock-transfected cell level, respectively (both P < 0.01) Consequently, expression of SLex, the product of a1,3-FucT-VII, was also elevated on the cell surface, to
171 and 284% of the mock-transfection value in FucTVII-M and FucTVII-H cells, respectively (both
P < 0.01; Fig 1C,D)
Expression of InR-a, EGFR and their SLexin a1,3-FucT-VII-transfected H7721 cells Expression of cell-surface InR-a and EGFR were ana-lyzed using specific antibodies and flow cytometry The
0 0.4 0.8 1.2
Mock FucTVII-M FucTVII-H
Mock FucTVII-M FucTVII-H
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FucTVII-M FucTVII-H
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*
0 40 80 120
160
*
*
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D C
Mock FucTVII-M
FucTVII-M FucTVII-H
FucT-VII
β-actin
FucTVII-H
497 bp
789 bp
10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
FL1-H
M1 M1
Fig 1 Characterization of VII cDNA-transfected H7721 cells (A) RT-PCR profiles of VII mRNA in mock- and a1,3-FucT-VII-transfected cells (B) Relative expressions of a1,3-FucT-VII mRNA in mock- and a1,3-FucT-VII transfected cells (n ¼ 3) (C) Fluorescence-activated cell spectra of cell-surface SLexon mock- and a1,3-FucT-VII transfected cell lines (D) Relative expressions of surface SLex on mock- and a1,3-FucT-VII transfected cells (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII (A) and (C) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ RT-PCR and flow cytometry are described in the Experimental procedures.
Trang 4results in Fig 2A,B show that their expression was not
obviously changed in a1,3-FucT-VII-transfected cells
compared with mock-transfected cells Results from
western immunoblots indicated that protein expression
in InR-a and EGFR was also unchanged following
transfection with a1,3-FucT-VII (Fig 2C) However,
after immunoprecipitation and western blotting of
these receptors, and using KM93 as the probe for
SLex, it was found that expression of SLex on InR-a
of FucTVII-M and FucTVII-H cells was increased to
248 and 409% of the mock-transfection level,
respect-ively (both P < 0.01), whereas expression of SLex on
EGFR remained unchanged (Fig 2D,E)
Tyrosine phosphorylation of InR-b, EGFR and IRS-1 in a1,3-FucT-VII-transfected H7721 cells
As shown in Fig 3A,B, the amount of immuno-precipitated InR-b was also unchanged following a1,3-FucT-VII transfection The relative intensity of tyrosine autophosphorylation in immunoprecipatated InR-b or EGFR was calculated from the intensity ratio of the phosphorylated band to the unphos-phorylated band Figure 3B also shows that tyrosine autophosphorylation of InR-b was increased to 186 and 352% of the mock-transfection value in FucTVII-M and FucTVII-H cells, respectively
0 30 60 90 120 150
EGFR InR
Mock FucTVII-M FucTVII-H
100 101 102 103 104 FL1-H M1
( - ) Control-InR ( - ) Control-EGFR
FucTVII-H-InR
0 50 100 150 200 250 300 350 400 450 500
EGFR EGFR SLeX
Mock FucTVII-M FucTVII-H
InR-α InR- α SLeX
E C
InR α
EGFR
β-actin
FucTVII-M-InR Mock-InR
FucTVII-H-EGFR FucTVII-M-EGFR
Mock-EGFR
10 0 10 1 10 2 10 3 10 4
FL1-H
M1
10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
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10 0 10 1 10 2 10 3 10 4
FL1-H
10 0 10 1 10 2 10 3 10 4
FL1-H M1
100 101 102 103 104 FL1-H M1
M1
M1
IP: InR- α WB: SLeX
WB: SLeX IP: EGFR
WB: EGFR IP: EGFR
IP: InR-α WB: InR-α
FucTVII-M FucTVII-H
Fig 2 Effects of a1,3-FucT-VII transfection on expression of InR-a and EGFR and the SLe x content of the glycans of InR-a and EGFR (A) Fluorescence-activated cell spectra of InR-a and EGFR on the cell surface (B) Relative expression of surface InR-a and EGFR (n ¼ 3) (C) Western immunoblot profiles of InR-a and EGFR after staining with an antibody to InR-a or EGFR and horseradish peroxidase-labeled sec-ondary antibody (D) Western immunoblot profiles of immunoprecipited InR-a and EGFR (precipitated by CF4) after staining with antibody to SLe x (KM93), InR-a or EGFR (antibody 528) and horseradish peroxidase-labeled secondary antibody to determine the SLe x content of InR-a and EGFR (E) Densitometric quantification of (D) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; InR, insulin receptor a-subunit; EGFR: epidermal growth factor receptor; IP, immunoprecipitation by the antibody to the protein indicated at the right; WB, western immunoblot with the antibody to the compound indicated at the right (A), (C) and (D) are representative of three repro-ducible experiments *P < 0.01 compared with ‘Mock’ Flow cytometry, immunoprecipitation and western immunoblot are described in the Experimental procedures.
Trang 5(both P< 0.01), whereas that of EGFR was
unchanged
Tyrosine phosphorylation of IRS-1 occurs earlier in
insulin signaling The IRS-1 protein was decreased in
a1,3-FucT-VII-transfected cells, although the change
was not statistically significant When relative tyrosine
phosphorylation was calculated as above, it was found
that the level of phosphorylated IRS-1 was increased
to 2.8 and 8.5 times that of the mock-transfection
value in FucTVII-M and FucTVII-H cells, respectively
(both P < 0.01; Fig 3C,D)
Phosphorylation and activity of PKB, expression
of PDK-1, PKN and phospho-PKN in
a1,3-FucT-VII-transfected H7721 cells
In insulin signaling, activation of PKB has been
impli-cated as a key step and it also has a major role in the
physiological effects of insulin [25] As shown in
Fig 4A,B, expression of PKB protein was not
obvi-ously altered in a1,3-FucT-VII-transfected H7721 cells,
but relative phosphorylation at T308 and S473 in PKB
(calculated from the ratio of the staining intensity of
phosphorylated protein to unphosphorylated protein
after normalization with b-actin) was apparently
eleva-ted when compared with mock-transfeceleva-ted cells After
densitometric quantification, relative T308
phosphory-lation was 149 and 205% of the mock-transfection
level in FucTVII-M and FucTVII-H cells, respectively (both P < 0.01) Meanwhile, relative S473 phosphory-lation was 170 and 315% of the mock-transfection value, respectively (both P < 0.01) Increased phos-phorylation of PKB at both T308 and S473 resulted in
an upregulation of PKB activity, measured as the amount of phosphorylated GSK3a⁄ b product As indi-cated in Fig 4C,D, phosphorylated GSK3a⁄ b was ele-vated to 165 and 270% of the mock-transfected value
in FucTVII-M and FucTVII-H cells, respectively (both
P < 0.01)
PDK-1 is the enzyme responsible for PKB phosphory-tion [26], and PKN is another substrate of PDK-1 rela-ted to cytoskeleton and transcription factor [27] Figure 4E,F shows that the PDK-1 protein was upregu-lated to 162 and 198% of the control value in
FucTVII-M and FucTVII-H cells, respectively (both P < 0.01) Protein expression of PKN and Ser⁄ Thr phosphoryla-tion of PKN (p-PKN) were also increased to a similar degree Therefore, the relative phosphorylation of p-PKN (pPKN⁄ PKN protein) was generally unchanged
Expression and phosphorylation of c-Raf-1, MEK and p42⁄ 44 MAPK in a1,3-FucT-VII-transfected H7721 cells
The Ras–Raf–MEK–MAPK pathway is another important signaling pathway in addition to the
0 50 100 150 200 250 300 350 400 450
k c M M -V T c u H -V T c u
InR-β InR-β-Tyr-p EGFR EGFR-Tyr-p
D
0 100 200 300 400 500 600
IRS-Tyr p IRS-1
k M M -V c F H -V c F
Mock FucTVII-M FucTVII-H IP: InR- β
WB: PT66
C
Mock FucTVII-M FucTVII-H IP: IRS-1
WB: PT66
IP: IRS-1 WB: IRS-1
WB: PT66 IP: EGFR
WB: EGFR IP: EGFR
IP: InR- β WB: InR- β
Fig 3 Effects of a1,3-FucT-VII transfection on the tyrosine autophosphorylation of two receptors and of IRS-1 (A) Western immunoblot pro-files of immunoprecipitated InR-b and EGFR (precipitated by CF4) after staining with antibody to phosphotyrosine (PT66), InR-b or EGFR (CF4) and horseradish peroxidase-labeled secondary antibody (B) Densitometric quantification of (A) (n ¼ 3) (C) Western immunoblot pro-files of immunoprecipitated IRS-1 after staining with phosphotyrosine antibody (PT66), IRS-1 antibody and horseradish peroxidase-labeled secondary antibody (D) Densitometric quantification of (C) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII;
WB, western immunoblot with the antibody to phosphotyrosine (PT66) or to the protein indicated on the right; IRS-1, insulin receptor sub-strate-1; Tyr-p, tyrosine phosphorylated (A) and (C) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Immunoprecipitation and western immunoblotting are described in the Experimental procedures.
Trang 6PDK-1⁄ PKB pathway in the insulin receptor [23]
Fig-ure 5A,B shows that expression of MEK and p42⁄ 44
MAPK proteins was not apparently altered in a1,
3-FucT-VII-transfected H7721 cells However,
expres-sion of c-Raf-1 increased significantly following
a1,3-FucT-VII transfection, being 168 and 325% of the
mock-transfection value in M and
FucTVII-H cells, respectively (both P < 0.01) Moreover, the
relative phosphorylation of MEK, as determined by
the ratio of p-MEK to MEK, was upregulated to 207
and 425% in FucTVII-M and FucTVII-H cells,
respec-tively (both P < 0.01), and the relative
phosphoryla-tion of p42⁄ 44 MAPK (the ratio of p-p42 ⁄ 44 MAPK
to p42⁄ 44 MAPK) was also increased in FucTVII-M
and FucTVII-H cells, being 2.82 and 6.01 times the
mock-transfection value (both P < 0.01)
Effect of HNMPA-(AM)3and KM93 on the
phosphorylation of PKB and p42/44 MAPK
In order to study whether the alteration in the
phos-phorylation of PKB and p42⁄ 44 MAPK was mediated
by InR kinase and surface SLex, phosphorylation of
these two signaling molecules was determined before
and after cultured cells were treated with 50 lm
HNMPA-(AM)3 (a specific inhibitor of InR tyrosine
kinase) [28] or 30 lgÆmL)1 KM93 (SLex antibody) for
24 h; corresponding untreated cells were used as the
control It was found that the results from the untreated cells were the same as those shown in Figs 4 and 5 When H7721 cells were treated with HNMPA-(AM)3, phosphorylation of PKB at both T308 and S473, and of p42⁄ 44 MAPK was apparently decreased in mock- and a1,3-FucT-VII- transfected cells (Fig 6A) The decrease
in phosphorylation of PKB and p42⁄ 44 MAPK was
~ 40.9–76.5% (P < 0.01) in a1,3-FucT-VII-transfected cells, compared with the corresponding untreated cells (Fig 6B) By contrast, differences in phosphorylation intensity for PKB and MAPK among mock,
FucTVII-M and FucTVII-H cell groups were attenuated in HNMPA-(AM)3-treated cells (Fig 6 A,B) Similarly, in the presence of KM93, phosphorylation of both PKB and p42⁄ 44 MAPK, and the differences in their phos-phorylation intensities among the three cell lines were also decreased significantly (Fig 6C,D) The reduction
in phosphorylation of PKB and p42⁄ 44 MAPK in a1,3-FucT-VII-transfected cells was 41.1–89.7% (P < 0.01) In the presence of HNMPA-(AM)3 or KM93, the rate of inhibition of phosphorylation was correlated with expression of a1,3-FucT-VII, which was FucTVII-H > FucTVII-H > mock-transfected cells However, some differences in the phosphorylation intensities of PKB and MAPK were observed in mock-and a1,3-FucT-VII-transfected cells in the presence of both inhibitors, but the differences were either not sta-tistically significant or P < 0.05
D
E
F
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0 100 200 300 400 500 600
PDK-1 PKN p-PKN
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PKB-T308
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Phosphorylated GSK3 α/β β-actin
FucTVII-M FucTVII-H
Mock PDK-1
PKN p-PKN
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FucTVII-M FucTVII-H
Fig 4 Effects of a1,3-FucT-VII transfection on the phosphorylation, protein expression and activity of some signaling molecules (A) Western blot profiles of PKB and T308-, S473-phosphorylated PKB after staining with specific antibodies and horseradish peroxidase-labeled sec-ondary antibody (B) Densitometric quantification of (A) (n ¼ 3) (C) Determination of PKB activity as the amount of phosphorylated GSK3a ⁄ b product (D) Densitometric quantification of (C) (n ¼ 3) (E) Western immunoblot profiles of PDK-1, PKN and p-PKN after staining with speci-fic antibodies and horseradish peroxidase-labeled secondary antibody (F) Densitometric quantispeci-fication of E (n ¼ 3) Mock, H7721 cells trans-fected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transtrans-fected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphorylated PKB at S473 (A), (C) and (E) are representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Western blotting, western immunoblot-ting and assay of PKB activity are described in the Experimental procedures.
Trang 7Expression of b-catenin and TCF in
a1,3-FucT-VII-transfected H7721 cells
Beta-catenin is a substrate of GSK-3 and a key
mole-cule in the Wnt and TGF-b signaling pathways [29–
31] As shown in Fig 7A,B, the level of b-catenin was
upregulated in a1,3-FucT-VII-transfected cells, to
242% in FucTVII-M cells and 504% in FucTVII-H
cells (both P < 0.01)
Luciferase activity was measured as an indicator of
the activity of transcription factor TCF As shown in
Fig 7C, TCF activity was also increased in
FucTVII-M and FucTVII-H cells, being 239 and 333% of the
mock-transfection level, respectively (both P < 0.01)
Modification of insulin signaling in a1,3-FucT-VII-transfected cells The effect of the above-mentioned changes in insulin-signaling molecules on transduction of the insulin sig-nal was further studied in a1,3-FucT-VII-transfected cells following serum starvation Phosphorylation at T308 and S473 of PKB and p42⁄ 44 MAPK was also selected as an indicator of signaling efficiency It was found that phospho-PKB-S473 was barely seen in insulin-untreated and serum-starved cells, but was expressed in insulin-treated cells By contrast, phospho-PKB-T308 and phospho-p42⁄ 44 MAPK were expres-sed in both insulin-untreated and insulin-treated cells The intensity levels for phospho-PKB-T308 and phos-pho-MAPK in both insulin-untreated and -treated cells, as well as the phospho-PKB-S473 in insulin-trea-ted cells, were FucTVII-H > FucTVII-M > mock (Fig 8A) In the presence of insulin, phosphorylation
of PKB and MAPK was obviously upregulated, and was significantly higher than in the corresponding control cells cultured in the absence of insulin The response to insulin stimulation was proportional to the expression of a1,3-FucT-VII In insulin-stimulated FucTVII-M and FucTVII-H cells, phospho-PKB-T308 was upregulated to 215 and 398% of the mock-transfc-tion level (both P < 0.01), and phospho-PKB-S473 was upregulated to 192 and 354% of the mock-trans-fection level, respectively (both P < 0.01) Similarly, phospho-MAPK was 184 and 345% of the mock-transfection level, respectively (both P< 0.01) (Fig 8B)
Discussion
The results shown in Fig 1A,B indicate that two a1,3-FucT-VII-transfected cell lines were established with moderate and high expression of the exogenous cDNA Expression of SLex, the product of FucT-VII, was positively correlated with expression of a1,3-FucT-VII mRNA (Fig 1C,D)
After transfection of the a1,3-FucT-VII cDNA, pro-tein expression of InR (including the a- and b-sub-units) and EGFR both on the cell surface and in cells (Fig 2A,C), as well as the SLex content of the glycans
of EGFR were unchanged, but the SLex content of the glycans of InR-a was increased significantly (Fig 2D,E) If the SLex of InR-a glycans is compared with that of EGFR in mock-transfected cells, it is observed that the SLexcontent of EGFR is far greater than that of InR-a (Fig 2D), suggesting that the SLex
on EGFR is high enough and cannot be upregulated further by overexpression of a1,3-FucT-VII This may
A
0
0
2
0
4
0
6
0
8
4 / 2 p K E M f
a
R
-c
K P A M
4 / 2 p -p K E M -p
K P A M
k c o M B
M -I V T c u F H -I V T c u F
Mock
c-Raf
p-MEK
MEK
p-p42/44
MAPK
p42/44
MAPK
β-actin
Fig 5 Effects of a1,3-FucT-VII transfection on the expression of
c-Raf-1 and phosphorylation of MEK and p42 ⁄ 44 MAPK (A)
West-ern immunoblot profiles of c-Raf-1, p-MEK, MEK, p-p42 ⁄ 44 MAPK
and p42 ⁄ 44 MAPK after staining with specific antibodies and
horse-radish peroxidase-labeled second antibody (B) Densitometric
quan-tification of (A) (n ¼ 3) Mock, H7721 cells transfected with
pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate
expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line
with high expression of transfected a1,3-FucT-VII; p-MEK,
phos-phorylated MEK; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK.
(A) is representative of three reproducible experiments *P < 0.01
compared with ‘Mock’ Western immunoblotting is described in the
Experimental procedures.
Trang 8be one reasons why a1,3-FucT-VII did not increase
the amount of SLexon EGFR Alternatively, the
com-position and structure of EGFR glycans probably
dif-fer from those of InR-a, and the EGFR glycans are
not suitable substrates for fucosylation by exogenous
a1,3-FucT-VII In other words, SLex on EGFR is
probably not synthesized by a1,3-FucT-VII, but by
other a1,3-FucTs
Our findings showed that transfection of
a1,3-FucT-VII promoted the functional activity of InR, as verified
by increased tyrosine phosphorylation of InR-b and
IRS-1 (Fig 3) Moreover, Ser⁄ Thr phosphorylation of
InR signaling molecules, including PKB (Fig 4A,B),
MEK, p42⁄ 44 MAPK (Fig 5A,B) and the activity of
PKB (Fig 4C,D) was stimulated concomitantly
Expression of some other signaling proteins, such as
PDK-1, PKN (Fig 4E,F), c-Raf-1 (Fig 5A,B) and
b-catenin (Fig 7A,B), was also upregulated by
a1,3-FucT-VII Elevation of Ser⁄ Thr phosphorylation in
downstream signaling molecules was presumed to be
mediated by increased tyrosine phosphorylation of InR
and IRS-1; the latter resulting from the increased SLex
content of InR-a This speculation was evidenced by
the following First, the intensity of Ser⁄ Thr
phos-phorylation in downstream signaling molecules was positively correlated with the intensity of tyrosine phosphorylation in InR and IRS-1, and tyrosine phos-phorylation was proportional to the SLex content of InR-a and also the mRNA expression of a-1,3-FucT-VII in mock, FucTa-1,3-FucT-VII-M and FucTa-1,3-FucT-VII-H cells Sec-ond, inhibition of InR tyrosine autophosphorylation
by HNMPA-(AM)3, which inhibits EGFR tyrosine kinase slightly [28], led to a dramatic reduction in the Ser⁄ Thr phosphorylation of PKB and p42 ⁄ 44 MAPK, and obvious attenuation of the difference in phos-phorylation intensity among three cell lines with differ-ent a1,3-FucT-VII expression levels (Fig 6A) Third, blockage of cell surface SLexby KM93 also resulted in significant attenuation of the phosphorylation of PKB and p42⁄ 44 MAPK, as well as the difference in phos-phorylation intensity among three cell lines (Fig 6B) However, in the presence of HNMPA-(AM)3 and KM93, some differences in the phosphorylation inten-sities of PKB and p42⁄ 44 MAPK were still observed
in mock- and a1,3-FucT-VII-transfected cells, indica-ting that the SLex on InR contributes a large propor-tion, though not all, of the increased phosphorylation caused by the overexpression of a1,3-FucT-VII It
0 0 0 0 0 0 0 700
3 ) M A ( A P M N H h t W 3
) M A ( A P M N H t u h t W
8 T -B K P 3 S -B K P K P A M 4 / 2 -p
*
#
#
0 0 0 0 0 0 0 0 0
3 M K h ti W 3
M K t u h ti W
p 3 T -B K P p 4 -B K P K P A M 4 / 2 -p
*
*
# #
# #
Mock-1
FucTVII-M
( ) HNMPA-(AM)3
PKB-T308p
PKB-S473p
p-p42/44 MAPK
β-actin
(+) HNMPA-(AM)3 FucTVII-H
Mock-1 FucTVII-M FucTVII-H Mock-2 FucTVII-M FucTVII-H
Mock-1 FucTVII-M FucTVII-H Mock-2 FucTVII-M FucTVII-H
Mock-2 FucTVII-M FucTVII-H
(+) KM93 ( ) KM93
FucTVII-H Mock-1 FucTVII-M Mock-2 FucTVII-M FucTVII-H
PKB-T308p
PKB-S473p
p-p42/44 MAPK
β-actin
Fig 6 Effect of HNMPA-(AM)3and KM93 on the phosphorylation of PKB and p42 ⁄ 44 MAPK (A) Western immunoblot profiles of phosphor-ylated PKB and p42 ⁄ 44 MAPK in the absence and presence of HNMPA-(AM) 3 (B) Densitometric quantification of (A) (n ¼ 3) (C) Western immunoblot profiles of phosphorylated PKB and p42 ⁄ 44 MAPK in the absence and presence of KM93 (D) Densitometric quantification of (C) (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphorylated PKB at S473; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK; Mock-1, mock cells without HNMPA-(AM) 3 or KM93 treat-ment; Mock-2, mock cells with HNMPA-(AM)3 or KM93 treatment (A) and (C) are representative of three reproducible experiments.
*P < 0.01 compared with ‘Mock-1’ #P < 0.05 compared with ‘Mock-2 Western immunoblotting is described in the Experimental proce-dures Samples without and with HNMPA-(AM) 3 or KM93 were examined simultaneously on the same electrophoresis gel.
Trang 9appears that upregulation of phosphorylation and
pro-tein expression was not mediated by EGFR, because
the SLex content and tyrosine autophosphorylation of
EGFR remained constant following a1,3-FucT-VII
transfection
In a previous insulin stimulation experiment, it was
found that H7721 cells were prone to die in
free (0%) medium; therefore, 2% fetal bovine
serum-deficient medium was used The results showed that
phospho-PKB-S473 barely appeared in cells cultured
in the serum-deficient medium (Fig 8A) As shown
in Fig 4A, however, there was basal expression of
both phospho-PKB-T308 and phospho-PKB-S473 in
mock- and a1,3-FucT-VII-transfected cells These observations suggest that phospho-PKB-T308 and phospho-PKB-S473 are regulated by different mechanisms It has been documented that phospho-PKB-T308 is regulated by phosphatidyl inositol-3-kinase⁄ PDK-1 [25,26], but the signal for PKB-S473 phosphorylation comes from the integrin⁄ integrin-linked kinase signaling pathway [32] Sarbassov et al reported that PKB-S473 can be phosphorylated directly by a kinase, named target of rapamycin (TOR) kinase and its associated protein rictor,
0 0 0 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4
K P A M 4 / 2 p -p p 7 S -B K P p 0 T -B K P
k c o M M -7 T H -7 T
*
*
*
*
*
*
Mock
A
B
FT7M
FT7H Mock FT7M FT7H
PKB-T308p
PKB-S473p
p-p42/44 MAPK
β-actin
Fig 8 Facilitation of insulin signaling in a1,3-FucT-VII-transfected cells (A) Western profiles of phosphorylated PKB and p22 ⁄ 24 MAPK in insulin-untreated and -treated cells cultured in 2% fetal bovine serum medium (B) Quantification of phosphorylated PKB and p22 ⁄ 24 MAPK in the presence of insulin (n ¼ 3) Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected a1,3-FucT-VII; PKB-T308p, phosphorylated PKB at T308; PKB-S473p, phosphoryl-ated PKB at S473; p-p42 ⁄ 44 MAPK, phosphorylated p42 ⁄ 44 MAPK; FT7M, H7721 cell line with moderate expression of transfected a1,3-FucT-VII; FT7H, H7721 cell line with high expression of trans-fected a1,3-FucT-VII (A) is representative of three reproducible experiments *P < 0.01 compared with ‘Mock’ Cell culture, insulin treatment and western immunoblotting are described in the Experimental procedures Samples without and with insulin treat-ment were examined simultaneously on the same electrophoresis gel.
0
200
400
600
A
B
C
*
*
0
5
10
15
20
25
30
*
*
β-actin
β-Catenin
Fig 7 Effects of a1,3-FucT-VII cDNA transfection on the
expres-sion of b-catenin and TCF activity (A) Western blot profile of
b-catenin (B) Densitometric quantification of (A) (n ¼ 3) (C)
Trans-activation activity of TCF measured as luciferase activity (n ¼ 3).
Mock, H7721 cells transfected with pcDNA3.1 vector; FucTVII-M,
H7721 cell line with moderate expression of transfected
a1,3-FucT-VII; FucTVII-H, H7721 cell line with high expression of transfected
a1,3-FucT-VII; TCF, T-cell factor (transcription factor) (A) is
repre-sentative of three reproducible experiments *P < 0.01 compared
with ‘Mock’ Western blotting and luciferase assay are described in
the Experimental procedures.
Trang 10because a reduction in rictor or mammalian TOR
(mTOR) expression inhibited the signaling of PKB
Rictor–mTOR complex can also facilitate the
phos-phorylation of PKB-T308 by PDK-1 [33] Basal
expression of phospho-PKB-S473 in serum-containing
medium may result from the stimulation of insulin or
growth factors in the 10% fetal bovine serum Our
finding that the cell response to insulin was
correla-ted with expression of a1,3-FucT-VII and SLex
(Fig 8) reveals that insulin signaling was facilitated
in a1,3-FucT-VII-transfected cells
Expression of PDK-1, PKN, c-Raf-1 and b-catenin
was upregulated after a1,3-FucT-VII transfection The
mechanism is not well understood However, it is
rea-sonable to speculate that upregulation may be caused
by the promotion of InR signaling, because activated
PKB can stimulate the phosphorylation of GSK-3 and
inhibit the activity of GSK-3 GSK-3 phosphorylates
b-catenin and stimulates the ubiquitination and
protea-somal proteolysis of b-catenin During activation of
the InR pathway, GSK-3 is inactivated, which leads to
the accumulation and nuclear translocation of
cyto-plasmic unphosphorylated b-catenin In nuclei,
b-cate-nin binds to transcription factor TCF (T-cell factor,
also called LEF, leukocyte enhancer factor) to form a
heterodimer, and transactivates the transcription of
target genes [29–31] This model of mechanism is
sup-ported by the increased activity of TCF in this study
(Fig 7C) Recently, we discovered that a1,3-FucT-VII
can upregulate expression of the integrin-a5 subunit at
both the mRNA and protein levels (unpublished) The
latter finding supports the suggestion that transfection
of a1,3-FucT-VII might affect the transcription of
some genes
It would be better to use antisense a1,3-FucT-VII,
iRNA or a gene-knockout method to suppress
endogenous a1,3-FucT-VII to confirm the above
results Unfortunately, we found that suppression of
a1,3-FucT-VII expression after transfection of
anti-sense a1,3-FucT-VII cDNA was not apparent,
because parent H7721 cells express a low level of
endogenous a-1,3-FucT-VII Sometimes antisense
cDNA even led to cell death When a gene of
a1,3-FucT-VII was knocked-out, almost all cells died
within 24 h This suggests a1,3FucT-VII is essential
for the survival of H7721 cells Therefore,
construc-tion of a plasmid containing a mutant at the
cata-lytic domain of a1,3-FucT-VII with deletion of
enzyme activity of its coding protein is very critical
if we are to determine whether the changed
phospho-rylation of signaling molecules was mediated by the
altered amount of SLex on InR This is being
inves-tigated in our laboratory
It would be of interest to study whether the SLex level of InR on insulin-responsive cells in diabetic patients was changed This may reveal the role of the sugar chains on InR in the pathogenesis of diabetes
In summary, the cDNA of a1,3-FucT-VII is able to regulate the phosphorylation and expression of some signaling molecules in the InR pathway, and these effects of a1,3-FucT-VII are probably mediated by its product, SLex, on the glycans of cell-surface receptors Increased expression and phosphorylation of insulin-signaling molecules leads to the facilitation of insulin signaling These findings provide evidence that modifi-cation of the terminal structure of glycans on surface receptors may also affect cell signaling The detailed mechanism requires further study
Experimental procedures
Materials
H7721 human hepatocarcinoma cell line was obtained from the Institute of Cell Biology RPMI-1640 and liposome
(Rock-ville, MD) Rabbit polyclonal antibodies against human insulin receptor a- and b-subunit, IRS-1, Raf-1, PDK-1, PKN, b-catenin and mouse mAb 528 (against human EGFR extracellular domain), b-actin and Protein G plus-agarose were from Santa Cruz Technology (Santa Cruz, CA) Rabbit polyclonal antibodies against human PKB,
(Beverly, MA) The PKB assay kit was from New England Biolabs (NEB Ltd., Beijing, China) KM93 was from Sei-kagaku Co (Tokyo, Japan) The InR kinase inhibitor,
CA) Polyvinylidene difluoride membrane was from Bio-Rad Laboratories (Hercules, CA) Phosphotyrosine anti-body (PT66), Mes, Hepes, leupeptin, pepstatin, human EGFR mAb CF4 (against intracellular domain), fluorescein isothiocyanate-conjugated and horseradish peroxidase-labe-led secondary antibodies (goat anti-mouse and anti-rabbit IgG) were from Sigma (St Luois, MO) Trizol, AMV reverse transcriptase, transcription factor TCF analysis kit
lucif-erase reporter plasmid (pRL-TK) were from Promega (Madison, WI) The TCF reporter plasmid (TK-luciferase reporter) was the product of Upstate Biotechnology (Lake Placid, NY) The RT-PCR primer of a1,3-FucT-VII was provided by TaKaRa Co (Tokyo, Japan) Other reagents were commercially available in China
H7721 cell lines were established as previously reported [15]