Glycobiology Advance Access originally published online on July 21, 2006
Glycobiology 2006 16(10):1007-1019; doi:10.1093/glycob/cwl023
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Down-regulation of trypsinogen expression is associated with growth retardation in 
1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity
3 Department of Glycotherapeutics, 4 Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0871; 5 Takara Bio Inc., Shiga 520-2193; 6 Department of Molecular Genetics, Kochi University Graduate School of Medicine, Kochi 783-8505, Japan; 7 CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012; and 8 the 21st century COE program, Ministry of Education, Culture, Sports, Science and Technology, 2-5-1 Marunouchi, Chiyoda-ku, Tokyo 100-8959, Japan
2 To whom correspondence should be addressed; e-mail:kondoa{at}glycot.med.osaka-u.ac.jp
Received on June 1, 2006; revised on July 3, 2006; accepted on July 10, 2006
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Alpha1,6-fucosyltransferase (Fut8) plays important roles in physiological and pathological conditions. Fut8-deficient (Fut8–/–) mice exhibit growth retardation, earlier postnatal death, and emphysema-like phenotype. To investigate the underlying molecular mechanism by which growth retardation occurs, we examined the mRNA expression levels of Fut8–/– embryos (18.5 days postcoitum [dpc]) using a cDNA microarray. The DNA microarray and real-time polymerase chain reaction (PCR) analysis showed that a group of genes, including trypsinogens 4, 7, 8, 11, 16, and 20, were down-regulated in Fut8–/– embryos. Consistently, the expression of trypsinogen proteins was found to be lower in Fut8–/– mice in the duodenum, small intestine, and pancreas. Trypsin, an active form of trypsinogen, regulates cell growth through a G-protein-coupled receptor, the proteinase-activated receptor 2 (PAR-2). In a cell culture system, a Fut8 knockdown mouse pancreatic acinar cell carcinoma, TGP49-Fut8-KDs, showed decreased growth rate, similar to that seen in Fut8–/– mice, and the decreased growth rate was rescued by the application of the PAR-2-activating peptide (SLIGRL-NH2). Moreover, epidermal growth factor (EGF)-induced receptor phosphorylation was attenuated in TGP49-Fut8-KDs, which was highly associated with a reduction of trypsinogens mRNA levels. The addition of exogenous EGF recovered c-fos, c-jun, and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, the EGF-induced up-regulation of c-fos and c-jun mRNA expression was significantly blocked by the protein kinase C (PKC) inhibitor. Our findings clearly demonstrate a relationship between Fut8 and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathway in controlling cell growth and that the EGFR-trypsin-PAR-2 pathway is suppressed in TGP49-Fut8-KDs as well as in Fut8–/– mice.
Key words:
cell growth
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FUT8 knockdown cell
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PAR-2
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trypsinogen
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1,6-fucosyltransferase
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Fut8 transfers fucose to the innermost GlcNAc residue of complex N-glycans via
1,6-linkage (core fucosylation) in the Golgi apparatus in mammals (Wilson et al., 1976
). Fut8-deficient (Fut8–/–) mice showed a dramatically changed phenotype similar to that of GDP-4-keto-6-deoxy-D-mannose epimerase–reductase (FX)-deficient (FX–/–) mice (Smith et al., 2002), showing a postnatal failure to thrive, and all the survivors manifested growth retardation (Wang et al., 2005). In comparison with other terminal fucose-deficient mice such as Fut1–/– (Domino et al., 2001), Fut2–/– (Domino et al., 2001), Fut7–/– (Maly et al., 1996), double Fut4–/–/Fut7–/– (Homeister et al., 2001), and Fut9–/– (Kudo et al., 2004) mice, which develop normally and exhibit no gross phenotypic abnormalities, the phenotypes of the Fut8–/– mice were much severe in terms of development and growth.
It is well known that trypsinogen is synthesized by pancreas acinar cells and is secreted into the intestinal lumen after the ingestion of food, where it is processed to active trypsin by enterokinase. Although the function of trypsin is traditionally related to the digestion of consumed proteins, recent discoveries suggest that it also serves as an activator of the proteinase-activated receptor 2 (PAR-2), which, in turn, induces various biological effects, such as cell growth (Ossovskaya and Bunnett, 2004). Four members of the PAR family have been cloned to date: PAR-1, PAR-3, and PAR-4 can be activated by thrombin, whereas PAR-2 is activated by trypsin and mast cell tryptase but not by thrombin (Kahn et al., 1998). PAR-2 is a seven transmembrane spanning G-protein-coupled receptor (Nystedt et al., 1994) and is widely expressed in several tissues and cell lines. Digestive organs, such as pancreas and intestine, especially highly express PAR-2 (Bohm et al., 1996; Ossovskaya and Bunnett, 2004). It has been reported that PAR-2 plays multiple physiological roles in various tissues such as ion transport, intestinal mobility, several exocrine secretion, and in the response of tissues to injury, including inflammation, pain, and healing (Ossovskaya and Bunnett, 2004). Indeed, 20% of all PAR-2–/– mice were either stillborn or died within 48 h of birth (Damiano et al., 1999). Trypsin could cleave the extracellular N-terminus of mouse PAR-2, at 33SKGR*SLIGRL42, exposing the tethered ligand domain SLIGRL, which then binds to conserved regions in the extracellular loop II of the cleaved receptor and triggers receptor function under physiological conditions (Nystedt et al., 1994). A synthetic peptide corresponding to the tethered ligand sequences (SLIGRL) can also activate PAR-2 selectively and is an important tool for studying the functions of mouse PAR-2. Indeed, either trypsin or SLIGRL-NH2 can stimulate the mitogen-activated protein kinase (MAPK) cascade and may regulate cell proliferation (Darmoul et al., 2004; Ossovskaya and Bunnett, 2004).
The epidermal growth factor receptor (EGFR) consists of an extracellular domain, a hydrophobic transmembrane domain, and an intracellular domain (Carpenter and Cohen, 1990). It has been reported that glycosylation is a common posttranslational feature in EGFR, which contains 11 N-glycosylation sites but no O-linked oligosaccharides (Stroop et al., 2000). Several researchers have reported that the N-glycosylation of EGFR is an important parameter in its ligand-binding activity (Pratt and Pastan, 1978; Soderquist and Carpenter, 1984; Rebbaa et al., 1997; Tsuda et al., 2000; Guo et al., 2004). Moreover, Stroop and others (2000) reported that the complex-type oligosaccharides linked to EGFR have di-, tri-, and tetra-antennary structures with core fucosylation. Wang and others (2005) reported that the binding affinity of the transforming growth factor-ß (TGF-ß) toward TGF-ß type II receptor is diminished in Fut8–/– mouse embryonic fibroblasts as well as in Fut8–/– mice. It is conceivable that core fucose residues of N-glycans associated with EGFR may modify its receptor function, such as ligand–receptor binding, dimerization, and phosphorylation.
Because 70% of Fut8–/– mice showed an earlier postnatal death, sophisticated methods were needed to study the functions of Fut8. Short interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells (Elbashir et al., 2001). To determine the underlying mechanism of the growth retardation seen in Fut8–/– mice, we established a Fut8 knockdown cell line by a siRNA technique using mouse pancreatic acinar cell carcinoma (TGP49). The Fut8 knockdown cells provided a good model for studying relationships among Fut8, trypsinogens, and growth retardation. We demonstrate here that core fucosylation of N-glycans on EGFR regulates cell proliferation by EGFR-trypsin-PAR-2 signaling.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Trypsinogen genes were down-regulated in Fut8–/– mice
The DNA microarray enables the simultaneous measurement and comparison of the expression levels of thousands of genes and has great advantages in investigating complex gene expression patterns aimed at the discovery of novel molecular functions (Schena et al., 1995
). To elucidate the underlying mechanism of growth retardation, mRNA expression profiles of Fut8+/+ versus Fut8–/– embryos were compared using an IntelliGene II mouse DNA Chip. We found 37 genes that showed more than 2-fold changes in their expressions between Fut8+/+ and Fut8–/– embryos (18.5 days postcoitum [dpc]). Among these genes, 23 were up-regulated and 14 were down-regulated in Fut8–/– mice (Supplemental Table IA and B). It is particularly noteworthy that five trypsinogens 4, 7, 8, 11, and 16 were found in grouped genes, expressions of which were lower in Fut8–/– mice (Table I). Twenty mouse trypsinogen genes were located on chromosome 6, trypsinogens 1–20 (GeneBank accession number AE000663
[GenBank]
, AE000664
[GenBank]
, AE000665
[GenBank]
), of which 11 (trypsinogens 4, 5, 7, 8, 9, 10, 11, 12, 15, 16, and 20) are thought to be transcribed into RNA, seven (trypsinogens 1, 3, 13, 14, 17, 18, and 19) are pseudogenes, and two (trypsinogens 2 and 6) are relic genes. The trypsinogens 11 and 8 corresponded to trypsin 3 and 4 on the DNA chip, respectively. The mRNA expressions of trypsinogens 8, 11, and 16 on this DNA chip showed 3.2-, 4.6-, and 5.6-fold reductions, respectively. The trypsinogens 4 and 7 corresponded to RIKEN cDNA 1810009J06 gene (NM_023707
[GenBank]
) and RIKEN cDNA 2210010C04 gene (NM_023333
[GenBank]
) on this DNA chip, respectively. The mRNA expression of trypsinogens 4 and 7 showed 3.6- and 7.0-fold reduction in Fut8–/– mice, respectively. The microarray data were then validated by real-time polymerase chain reaction (PCR). The expression levels of several trypsinogens were similar to those observed in the DNA microarry analysis (Table I). Moreover, the expression of trypsinogen 20 (mouse pancreatic trypsin), which is thought to be involved in food digestion, proteolysis, peptidolysis, and hydrolytic activity in the mouse, showed a 4.9-fold reduction by the specific real-time PCR primers for trypsinogen 20 (Table II). Indeed, the cDNA sequence of trypsinogen 20 shared a high degree of identity (67, 74, 91, 88, and 89%) with trypsinogens 4, 7, 8, 11, and 16, respectively.
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Trypsinogen proteins in tissue homogenates from the duodenum, small intestine, and pancreas were detected by western blot analysis. The expression of the trypsinogens was significantly lower in Fut8–/– mice than in Fut8+/+ mice (Figure 1A). The trypsinogens bands represented the autolytically cleaved so-called two-chain form. In addition, gelatin zymography also showed a reduction of trypsin in Fut8–/– mice (data not shown). Indeed, the pancreas tissues express core fucosylated N-glycans, as confirmed by fucose lectin, Aspergillus oryzae lectin (AOL). Immunostaining of the pancreas specimens with anti-trypsinogen immunoglobulin (Ig)G also showed a lower expression of trypsinogens in Fut8–/– mice, compared with Fut8+/+ mice (Figure 1B). Collectively, these results suggest that the core fucosylation of N-glycans correlates with trypsinogen expressions and gene deletion of Fut8 results in the reduction of trypsinogen expression at both the mRNA and protein levels.
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Loss of Fut8 induced the down-regulation of trypsinogen expression in TGP49 cells
To clarify the association of Fut8 with trypsinogen expression, we attempted to knockdown Fut8 using siRNA in TGP49 cells (Pettengill et al., 1994). First, we designed specific Fut8-siRNA fragments complementary to different regions of Fut8 Open Reading Frame to observe the knockdown efficiency of Fut8. The sequences of the siRNA correspond to nucleotides 1016–1034 (No. 2), 2302–2320 (No. 5), 918–936 (No.8), and 1386–1404 (No.16) of mouse Fut8 (NM_016893
[GenBank]
). Among the four siRNA fragments, siRNA (No. 16) was the strongest inhibitor of the Fut8 expression, suppressing about 50% of the FUT8 activity, and this effect was not long lasting (data not shown).
It is important to assess the role of Fut8 under conditions where the effect of endogenous Fut8 is completely eliminated. To stably silence Fut8 expression, we designed a retrovirus expression system using a pSINsi-mU6 cassette vector inserted with Fut8 siRNA (No. 16) and established stable Fut8 knockdown cell lines, referred to as TGP49-Fut8-KD1 and TGP49-Fut8-KD2. As shown in Figure 2A, B, and C, the introduction of Fut8 siRNA almost completely suppressed Fut8 expression. The Fut8 mRNA expressions were reduced to 4.9 and 2.1% of the control in TGP49-Fut8-KD1 and TGP49-Fut8-KD2, respectively (Figure 2A). No apparent changes were found in the expressions of other glycosyltransferase genes, such as N-acetylglucosaminyltransferase III (GnT III) and ß1, 4-galactosyltransferase I (ß4GalT-I), suggesting that there is no off-target effect in this system (Supplemental Figure 1). FUT8 activities were barely detectable in TGP49-Fut8-KD1 and TGP49-Fut8-KD2 (Figure 2B). If any, the FUT8 activities of TGP49-Fut8-KDs were less than 5% of those of TGP49-Fut8-WT (759.1 pmol/h/mg) as well. Again, an Aleuria aurantia lectin (AAL) blot analysis reflected the results of the mRNA expressions and enzyme activities (Figure 2C). In TGP49-Fut8-KDs, the expression of trypsinogen proteins and mRNA was also reduced (Figure 2D and E). A sequence analysis of the trypsinogen cDNA cut from the gel showed that its cDNA sequence completely matched with trypsinogen 20 in GeneBank (NM_009430 [GenBank] ) (data not shown). It is conceivable that introducing siRNA resulted in the elimination of Fut8 expression, followed by the down-regulation of trypsinogens expression in TGP49-Fut8-KDs.
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Fut8 ablation led to the reduced proliferation, and it is rescued by the application of PAR-2 agonist, SLIGRL-NH2
The growth rates of TGP49-Fut8-KDs were much slower than those of TGP49-Fut8-WT, resembling the growth retardation of Fut8–/– mice (Figure 3). Cell confluence was reached between days 2 and 3 for TGP49-Fut8-WT and between days 4 and 5 for TGP49-Fut8-KDs. Trypsin has been shown to play a role in regulating cell growth by interacting with PAR-2 in addition to its role in digestion. In pancreatic cancer cells, trypsinogens were reported to be activated into trypsin by urokinase-type plasminogen activator (uPA) (Uchima et al., 2003). We detected uPA in TGP49-derived cell culture supernatants by western blot and gelatin zymograms (Supplemental Figure 2 and data not shown). TGP49-Fut8-WT and TGP49-Fut8-KDs expressed comparable levels of PAR-2, as detected by western blots and immunocytochemical analysis (Figure 4A and B). Bands at about 55 kDa corresponding to the known molecular mass of PAR-2 were visualized by western blot analysis (Figure 4A). In addition, an analysis of PAR-2 localization by confocal microscopy demonstrated that the PAR-2 expression of TGP49-Fut8-KD1 was, similar to TGP49-Fut8-WT, localized mainly on the plasma membrane (Figure 4B). PAR-2 contains two sites for N-linked glycosylation at Asn30, in proximity to the activation site in the extracellular tail, and at Asn222 in the extracellular loop II of PAR-2. In contrast to TGP49-Fut8-WT, the core fucosylation of TGP49-Fut8-KDs was barely detectable by AOL staining (Figure 4C). These results suggest that the core fucosylation of PAR-2 does not affect its stability and cell surface expression.
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SLIGRL-NH2 is a well-known and useful probe for assessing the biological role of PAR-2. Shimamoto and others (2004) reported that SLIGRL-NH2 stimulates cell proliferation through PAR-2 in pancreatic cancer cells. Here, the effect of SLIGRL-NH2 treatment on cell growth was studied in TGP49-derivative cells. The proliferation of TGP49-Fut8-KDs was stimulated by 25 and 50 µg/mL of SLIGRL-NH2, and stimulation by 25 µg/mL of SLIGRL-NH2 evoked a cell growth rate comparable to the control level (Figure 5). The blockade of PAR-2 with 5 µg/mL of anti-PAR-2 antibody completely prevented SLIGRL-NH2-mediated cell proliferation, indicating the crucial role of PAR-2 in cell proliferation (Figure 5). Moreover, in comparison with TGP49-Fut8-KDs, 30
40% of the cell growth of TGP49-Fut8-WT was suppressed by application of 5 µg/mL of the anti-PAR-2 antibody (Figure 3), indicating that the growth of TGP49 cells is partially dependent on PAR-2 stimulation in an autocrine manner. These findings clearly show that the reduced trypsinogen in Fut8–/– mice and/or in TGP49-Fut8-KDs are directly associated with cell growth control, at least in part, through the PAR-2 pathway.
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Attenuation of EGF-induced EGFR phosphorylation led to down-regulation of trypsinogen expression
Because it has been reported that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces trypsinogen expression in endothelial cells (Koshikawa et al., 1997), we suspected that protein kinase C (PKC) signaling could be involved in the trypsinogen reduction in TGP49-Fut8-KDs. PKC is activated by many types of cell surface receptors such as growth factor receptor and G-protein-coupled receptor. EGFR was highly expressed in pancreatic tissue and has been implicated in PKC activation. Therefore, we investigated the issue of whether a difference exists in the acquisition of the ligand-induced EGFR activation between TGP49-Fut8-WT and TGP49-Fut8-KDs. As shown in Figure 6A, the core fucosylation on the N-glycans of EGFR was diminished in TGP49-Fut8-KDs as confirmed by AOL staining. In contrast to TGP49-Fut8-WT, EGF-induced EGFR phosphorylation was attenuated in TGP49-Fut8-KD1 without any effect on the total EGFR expression levels at 5 min after stimulation (Figure 6B). The kinetics of receptor phosphorylations was quite similar in both cell lines, reached to the maximal level at 5 min, and returned to the basal level within 30 min. In TGP49-Fut8-KD1, the extent of phosphorylation of the receptor was less than half that of TGP49-Fut8-WT (Figure 6C). Moreover, we explored the potential role of EGF-associated signaling during the induction of trypsinogen expression by real-time PCR. As anticipated, excess EGF (10 ng/mL) overcame the lower EGFR activity and induced a more than 2-fold higher trypsinogen expression in TGP49-Fut8-KD1, which were detectable at 3 and 6 h, as well as TPA (20 nM) stimulation (Figure 7). The c-fos and c-jun are well-known targets of EGF stimulation (Angel and Karin, 1991; Chen and Davis, 2003) and play a variety of roles in many cell functions, such as cell growth and differentiation. In TGP49-Fut8-KD1, their mRNA expressions were reduced to about 30% those of TGP49-Fut8-WT level. Both EGF (10, 50 ng/mL) and TPA (20, 100 nM) induced several-fold increases in c-fos and c-jun expression, which were detectable at 30 min (Figure 8). EGF-induced c-jun expression in TGP49-derived cells was completely blocked by pretreatment with 1 µM of bisindolylmaleimide (BIM); however, the c-fos expression was partially inhibited, indicating that EGF regulates c-fos and c-jun expression in PKC-dependent and PKC-independent manners (Figure 8). It is noteworthy that EGF-induced trypsinogen expression was preceded by the expression of these two transcription factors, suggesting that c-fos and c-jun are able to directly regulate trypsinogen expression. We also investigated the expression levels of c-fos and c-jun in Fut8–/– mice. As expected, the mRNA expression of c-fos and c-jun extracted from the bodies of Fut8–/– mice is only 35.4 and 57.4% of Fut8+/+ mice, respectively (Figure 9). Collectively, these findings therefore suggest that the loss of core fucose residues on N-glycans of EGFR attenuated cellular signaling, such as PKC activation, resulting in a reduction in trypsin-PAR-2-regulated growth stimulation.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Because Fut8 is able to modify multiple proteins and affect their functions, it is necessary to analyze global changes to study the complicated phenotypes of Fut8–/– mice. Gene expression profiling using DNA microarray analysis is a powerful and popular approach for the comprehensive analysis of gene expressions in an unbiased manner. Using a DNA microarray analysis, we identified reduced trypsinogen gene expression in Fut8–/– embryos (18.5 dpc) compared with Fut8+/+ embryos (Table I). Moreover, the protein expression of trypsinogens from the duodenum, small intestine, and pancreas in Fut8–/– mice (4 days old) was lower than that in Fut8+/+ mice (Figure 1A and B). Because trypsinogens (trypsin) play critical roles, not only in digestion (Stevenson et al., 1986
) but in tissue remodeling (Koshikawa et al., 1998) and acrosomal reactions (Ohmura et al., 1999), under physiological conditions, we speculate that the low level of trypsinogens in Fut8–/– mice results in malnutrition, which may, in turn, lead to growth retardation. On the contrary, trypsin, an active form of trypsinogen, is also known to be most potent agonist of the PAR-2. Trypsin activates PAR-2 signaling transduction, leading to the proliferation and differentiation of several types of cells such as epithelial and endothelial cells, fibroblasts, astrocytes, and myocytes (Darmoul et al., 2004; Ossovskaya and Bunnett, 2004). The application of SLIGRL-NH2 (25 µg/mL) rescued the lower growth rates accompanying the down-regulation of trypsinogens, returning them to normal levels in TGP49-Fut8-KDs (Figure 5). Moreover, the cell growth of TGP49-Fut8-WT was suppressed by the application of an anti-PAR-2 antibody, by up to 6070% (Figure 3). These findings strongly suggest that reduced PAR-2 signaling is directly associated with the slower growth rates seen in TGP49-Fut8-KDs.
Trypsinogens are ubiquitously expressed in pancreas, skin, esophagus, stomach, small intestine, large intestine, lung, kidney, liver, bile duck, spleen and brain, and in endothelial and epithelial cells (Koshikawa et al., 1998). In addition to pancreatic trypsin, extrapancreatic trypsin is able to cleave PAR-2 (Alm et al., 2000). PAR-2 is also ubiquitously distributed throughout the entire body of mammals, including the pancreas, kidney, gastrointestinal and respiratory tracts, skin, liver, and heart (Bohm et al., 1996). PAR-2 has been demonstrated to be present in the pancreas and in every region of the gastrointestinal tract and regulates pancreatic and gastric functions. In the pancreas, PAR-2 is expressed by both acinar cells (Bohm et al., 1996; Kawabata et al., 2002) and the duct epithelium (Nguyen et al., 1999), where its activation triggers amylase secretion and electrolyte transport, respectively. Likewise, in the gastrointestinal tract, PAR-2 is expressed at high levels in epithelial cells, and its activation mediates gastrointestinal motility (Corvera et al., 1997) and modulates ion transport (Vergnolle, 2000). Collectively, the widespread distribution of PAR-2 in the pancreas and gastrointestinal tract, coupled with the fact that the pancreas and gastrointestinal tract are exposed to trypsin under physiological conditions, suggests that PAR-2 activation through trypsin is an important modulator in the pancreas and gastrointestinal tract. In Fut8–/– mice, we confirmed that trypsinogen (trypsin) expression in the duodenum, small intestine, and pancreas was much lower than that in Fut8+/+ mice (Figure 1A and B). Thus, reduction of trypsinogen levels resulted in the growth retardation of Fut8–/– mice, at least in part, via an attenuation of PAR-2 signaling.
Various studies have reported a role for N-glycosylation of EGFR in the direct regulation of receptor functions, including membrane phosphorylation, ligand binding, and signal transduction (Soderquist and Carpenter, 1984). Although specifically how various structures of N-glycans modify EGFR function is not known at present, functional studies about EGFR in the overexpressed cells of GnT III, an enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue, showed that the EGFR displayed a reduced binding affinity for EGF (Rebbaa et al., 1997). Tsuda and others (2000) reported that the removal of N-glycans at the Asn420 site in EGFR causes a loss of capability to bind EGF, which results in autoactivation of the receptor. The oligosaccharides linked to EGFR have been thoroughly characterized by nuclear magnetic resonance (NMR) and mass spectrometry (Stroop et al., 2000). Over 30 structures have been identified to date, and the major complex-type oligosaccharides contain a di-, tri-, and tetra-antennary structure with core fucosylation. In the present study, we found that core fucose was involved in the phosphorylation of EGFR (Figure 6). In addition, Wang and others (2005, 2006) reported that the binding affinity of TGF-ß type II receptor and EGFR toward respective ligands is reduced in mouse embryonic fibroblasts derived from Fut8–/– mice, suggesting the involvement of core fucose in ligand binding and the subsequent activation of various receptors. It has been reported that the 1,6-fucosylation could affect the conformation and flexibility of the antenna of N-linked bi-antennary oligosaccharides (Stubbs et al., 1996). This might be the case in EGFR conformation. It is conceivable that the lack of core fucose might cause conformational changes in the receptor on the cell surface, subsequently affecting the ligand binding, dimerization, and phosphorylation of the EGFR.
EGFR is coupled to multiple signaling pathways and regulates a wide variety of cellular functions including cell proliferation and differentiation. Mice lacking EGFR show various abnormalities including early death, developmental defects in gastrointestinal organs, etc. (Miettinen et al., 1995). It is important to note that EGFR-deficient mice also show severe defects in the lung, similar to Fut8–/– mice (Miettinen et al., 1997). EGF induces c-fos and c-jun via extracellular signal-regulated kinase (ERK)-dependent and PKC-dependent manners in bovine luteal cells (Chen and Davis, 2003). In mammalian cells, ERK and jun N-terminal kinase (JNK) are involved in increasing the amount of c-fos and c-jun, respectively (Karin and Hunter, 1995). The JNK pathway stimulates c-jun activation, which requires the costimulation of both the calcium- and PKC-dependant pathways. Consistent with this, our data indicated that excess EGF induced several-fold increases in c-fos and c-jun expression, and the induced c-jun expression was completely blocked by pretreatment with BIM, but the induced c-fos expression was only partially blocked (Figure 8). Moreover, the level of trypsinogen expression was increased 3 h after the activation of either EGF or TPA. These results suggest that the attenuation of EGF-induced EGFR phosphorylation results in the down-regulation of trypsinogen transcription via the insufficient activation of c-jun and c-fos. It should be noted that the mRNA expression of c-fos and c-jun extracted from the bodies of Fut8–/– mice are only 35.4 and 57.4% that of Fut8+/+ mice, respectively (Figure 9), suggesting that the signal transduction initiated by several receptors located on the plasma membrane, like EGFR, might be compromised in Fut8–/– mice.
In summary, the loss of core fucose on the extracellular domain of EGFR affects cell proliferation by modulating EGFR phosphorylation. The low levels of c-fos and c-jun expression in Fut8–/– mice are associated with growth retardation, at least in part, by a reduced trypsinogen expression which, in turn, attenuates the PAR-2-medaited growth signal pathway. The important finding of our study was that core fucose on the extracellular domain of EGFR has an effect on cell proliferation through the regulation of EGFR-trypsin-PAR-2 signaling. Prior studies have shown that tumor-derived trypsin might promote cell proliferation by activation of PAR-2 in several cancers (Miyata et al., 2000; Okamoto et al., 2001; Jin et al., 2003; Ohta et al., 2003; Darmoul et al., 2004). In addition, PAR-2 expression is closely correlated with trypsin expression in tumor tissues (Yamashita et al., 2003). In this regard, our present work provides an attractive opportunity toward a more effective strategy for cancer therapy by de-core fucosylation using the siRNA technique of Fut8, as well as the elucidation of the physiological roles of Fut8.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Mice
Homozygous wild (Fut8+/+) and knockout (Fut8–/–) mice were obtained by crossing heterozygous Fut8+/– mice. Only male animals of Fut8+/+ and Fut8–/– mice were used in this experiment. Zfy primer: the forward primer 5'-AAGATA AGCTTACATAATCACATGGA-3' and the reverse primer 5'-CCTATGAAATCCTTTGCTGCACATGT-3' were used for PCR to distinguish male animals. The mice were maintained in a room illuminated for 12 h (08:00–20:00 h) and kept at 24 ± 1°C with free access to food and water.
Antibodies
The mouse monoclonal anti-PAR-2 IgG2a antibody (sc-13504), mouse monoclonal anti-ß-actin IgG1 antibody (sc-8432), rabbit polyclonal anti-uPA antibody (sc-14019), goat polyclonal anti-1-antitrypsin antibody (sc-14586), and biotin-conjugated anti-goat IgG (sc-2053) were purchased from Santa Cruz (Santa Cruz, CA); anti-rabbit IgG horseradish peroxidase (HRP)-conjugate and anti-mouse IgG HRP-conjugate were from ICN Pharmaceuticals, Inc. (Arora, OH); Alexa Fluor 488-conjugated anti-mouse IgG was from Invitrogen (Carlsbad, CA); anti-phosphotyrosine antibody (PY20) and anti-EGFR antibody were from BD Transduction Laboratories (San Jose, CA). Rabbit polyclonal anti-trypsinogen serum was obtained from Biogenesis (Kingston, NH), and an IgG fraction of the antiserum was purified by affinity chromatography on a HiTrap Protein G HP Columns (Amersham Bioscience, Piscataway, NJ). This antibody was raised against bovine pancreatic trypsinogens. According to the manufacture’s protocol, it could cross-react with trypsinogens from various species including mouse and human and several types of trypsinogens.
Tissue specimens and sample preparation
At 18.5 dpc, mother mice were sacrificed by cervical dislocation, and the embryos were quickly removed. Fut8+/+ and Fut8–/– embryonic biopsies were obtained from three mice (one per mouse) for microarray and real-time PCR. Several tissues (pancreas, duodenum, and small intestine) obtained from 4-day-old Fut8+/+ and Fut8–/– mice were immediately fixed in 4% paraformaldehyde for use in immunhistochemical analyses. For protein expression analysis, each tissue was homogenized in 5 volumes (W/V) of ice-cold buffer (50 mM Tris–HCl, 150 mM of NaCl, 1% Triton-100 and 1 mM EGTA) and cleared by centrifugation at 20,000 g for 10 min and stored at –80°C until used. The protein concentration of each sample was assayed using a BCA protein assay kit (Pierce, Rockford, IL) according to the manufacturer’s protocol.
Complementary DNA microarray analysis
Freshly dissected Fut8+/+ and Fut8–/– embryonic body (18.5 dpc) samples were immediately submerged in 5 volumes of RNAlater (Ambion Inc., Woodward Austin, TX). Total RNA was extracted with TRIzol reagent (Invitrogen), following the protocol recommended by the manufacturer. Approximately 2 µg of total RNA was obtained per mg of embryonic body. Messenger RNA was isolated using a µMACs mRNA Isolation Kit (Miltenyi Biotec., Bergisch Gladbach, Germany). The integrities of total RNA and mRNA were verified by agarose gel electrophoresis and Bioanalyzer with RNA LabChip (Agilent, Palo Alto, CA), following the manufacture’s instruction. Messenger RNAs from three independent experiments were pooled for microarray.
For microarray hybridization, each mRNA (200 ng) was converted to labeled cDNA using RNA Fluorescence Labeling Core Kit (TAKARA Bio. Inc., Shiga, Japan) in the presence of Cy3-dUTP or Cy5-dUTP (Amersham Bioscience), and the labeled cDNA was then purified with Purification Column (TAKARA Bio. Inc.). Each RNA sample was labeled either with Cy3 or with Cy5 for two analyses (i.e., Cy dye swapping). Analysis of gene expression was performed using Takara IntelliGene II Mouse Chip (TAKARA Bio. Inc.), which contains 4285 genes. The hybridizations to the microarray chip were conducted according to the instruction for the Takara DNA Chip Protocol. Fluorescent intensities of the printed cDNA targets were measured using a fluorometric scanning Affymetrix 428 Array Scanner. The microarray images were analyzed using ImaGene v4.1 software. Fold-change levels were calculated, and genes that were regulated with a fold change 
2 were then selected.
Quantitative gene expression analyses by real-time PCR and RT–PCR
Real-time PCR analyses were performed using a Smart Cycler II System (Cephied, Sunnyvale, CA). The cDNA synthesis was performed using SYBR Green Real-time PCR Core Kit (TAKARA Bio. Inc.) as recommended by manufacturer. Each reaction was performed in a 25 µL volume with final concentration of 1 x SYBR Premix Ex Taq, 200 nM primers, 2 µL of 1:10 dilution of the cDNA and RNase-free water. The thermal cycling conditions for the real-time PCR were 10 s at 95°C to activate SYBR Ex Taq, followed by 40 cycles of denaturation 5 s at 95 °C and annealing/extension for 20 s at 60°C. The mean number of cycles to the threshold of fluorescence detection was calculated for each sample, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was quantified to normalize the amount of cDNA in each sample. The specificity of the amplified products was monitored by its melting curve.
To determine the effects of EGF and TPA on induction of the expression of c-jun, c-fos, and trypsinogen mRNA, we quantified the amounts of mRNA expression by real-time PCR using SYBR Green Real-time PCR Core Kit (TAKARA Bio. Inc.). Cultures that grew to 80–90% confluence were washed three times with serum-free medium before exposure of either EGF or TPA. In studies involving the pharmacological inhibitor, BIM, the PKC inhibitor, was added to the cells for 60 min before stimuli. After cell stimulation, the cells were rapidly rinsed twice with cold phosphate-buffered saline (PBS), and total RNA was extracted for real-time PCR. The real-time PCR primers are shown in Table II.
Reverse transcription–PCR (RT–PCR) was performed according to the usual method. The primer sequences of trypsinogen 20 were as follows: the forward primer was 5'-TGTTGATTCTGCCAAGATCATC-3', the reverse primer was 5'-TGCAGCTCTCCATTGCAGACC-3'. Reaction conditions included 94°C for 30 s, 60°C for 1 min, and 72°C for 2 min.
Immunohistochemistry
Immunohistochemical staining was performed on paraformaldehyde-fixed paraffin sections. Briefly, sections were deparaffinized twice in xylene and hydrated through a graded series of ethanol to PBS. Endogenous peroxidase activity was blocked with 3% H2O2 for 5 min. After washing with PBS containing 0.1% Tween 20 (PBS-T), the slides were blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). The slides were incubated with either anti-trypsinogen IgG or biotin-conjugated AOL (Ishida et al., 2002) and washed three times with PBS-T. After washing, the antibody-incubated slide was treated with biotin-conjugated anti-rabbit IgG for 1 h. The slides were washed again and then covered with HRP-streptavidin (Vector Laboratories) for 30 min. Finally, the slides were visualized with 3, 3'-diaminobenzidine and counterstained with hematoxylin and eosin.
Measurement of FUT8 activity
The specific activity of FUT8 was determined as described previously (Uozumi et al., 1996). Briefly, a 5-µg sample as the enzyme source was mixed with the assay buffer (200 mM MES [pH 7.0], 1% Triton X-100, 500 µM donor [GDP-L-fucose], 50 µM acceptor [GnGn-Asn-4-(2-pyridylamine) butylamine {PABA}]). After incubation at 37°C, the reaction was stopped by boiling. Ten microliters of the supernatant was subjected to high-performance liquid chromatography (HPLC) with a Fluorescent detector (Ex. 310 nm and Em. 380 nm). Activity was expressed as pmol of GDP-fucose transferred to the acceptor per hour per milligram of protein.
Cells culture
The pancreatic acinar cell carcinoma TGP49, in which trypsinogen is expressed (Pettengill et al., 1994), was obtained from ATCC (Manassas, VA; CRL-2136) and maintained in DMEM:Ham’s F12 (1:1) medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM Glutamine, 50 units/mL of penicillin, and 50 µg/mL of streptomycin (Sigma, St. Louis, MO). The medium was changed every 3 days. The subculture was conducted by washing the cells with PBS and adding 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Nakalai tesque, Kyoto, Japan) for 5 min. Trypsinized cells were counted, centrifuged, and resuspended in fresh medium. Cell lysates were prepared by scraping cells with ice-cold buffer (50 mM Tris–HCl, 150 mM of NaCl, 1% Triton-X 100, and 1 mM EGTA), followed by sonication. The lysate was cleared by centrifugation at 20,000 ¥ g for 10 min at 4 °C.
Transient transfection of Fut8 siRNA
TGP49 cells (5 x 104) were seeded in six-well plates and allowed to grow to 90% confluence. Transient transfections of Fut8 siRNAs were performed with TransIT-TKO transfection reagent (TAKARA Bio. Inc.) according to the manufacturer’s instructions. The siRNAs were designed to form 19-bp dsRNA with 2 thymine overhangs at both 3' ends. Four targeting sequences of the Fut8 siRNA used are as follows: No.2 (sense: 5'-UGGAGCUAAAGAGCUCUGGTT-3', antisense: 3'-TTACCUCGAUUUCUCGAGACC-5'); No 5 (sense: 5'-CAGCUUGUUAAGGCCAAAGTT-3', antisense: 3'-TTGUCGAACAAUUCCGGUUUC-5'); No 8 (sense: 5'-CAGGCUUAUAUCCCUCCUATT-3', antisense: 3'-TTGUCCGAAUAUAGGGAGGAU-5'); and No 16 (sense: 5'-UCUCAGAAUUGGCGCUAUGTT-3', antisense: 3'-TTAGAGUCUUAACCGCGAUAC-5'). Alexa 488-conjugated siRNA duplex (Qiagen, Hilden, Germany) was used to define the transfection efficiency.
Establishment of Fut8 knockdown cell lines
A retrovirusvecor carrying siRNA targeted to Fut8 (No.16) was constructed as follows. A 21-nucleotide sequence of the Fut8 gene was inserted in the sense and antisense directions into the pSINsi-mU6 cassette vector (recombinant retrovirus vector) (TAKARA Bio. Inc.) containing the mouse U6 promoter. The recombinant retroviruses were generated by co-transfection of the vector mixture such as recombinant retrovirus vector, pE-eco vector (ecotropic env), and pGP vector (gag-pol) to HEK293 cells. Recombinant retrovirus particles containing the target sequence or a mock control were infected into the parental TGP49 cells, and the geneticin (G418)-resistant clones were selected as a stable tranfectant. The TGP49 derivative cell lines stably transfected with the plasmid-expressing siRNA that targeted FUT8 are referred to hereafter as "GP49-Fut8-KD"and with the empty plasmid as "GP49-Fut8-WT."
Cell growth assay
The growth rate of cells was measured using the Cell Counting Kit-8 (CCK-8) (Wako, Osaka, Japan). Each cell line was grown in 96-well tissue culture dishes at 37°C in DMEM:Ham’s F12 (1:1) medium containing 10% FBS and 200 µg/mLof G418. Ten microliters of CCK-8 solution to each well was used for measuring the number of living cells. The absorbance related to the formazan dye level was measured with a microplate reader (Corona Electric Co., Ibaraki, Japan) at 490 nm. To determine the proliferative effect of the PAR-2 agonists in the TGP 49 cell culture, the cells were treated with or without activating peptide SLIGRL-NH2 (25 and 50 µg/mL) (Sigma) in DMEM:Ham’s F12 (1:1) medium containing 2% bovine serum albumin (BSA). After an appropriate period of incubation, the rate of cell growth was assessed with a hemacytometer.
Fluorescence immunocytochemistry and confocal microscopy
For cellular localization of PAR-2 in TGP49 cells, each cell line was seeded on a FALCON and BIOCOAT culture slide (four chambers; Becton Dickinson Labware, Meylan Cedex, France). When the cells reached subconfluence, they were washed with PBS (PBS+: 0.9 mM CaCl2 and 0.5 mM MgCl2) and fixed with 4% paraformaldehyde. After washing with PBS, cells were blocked with 5% BSA in PBS+. Following a PBS rinse, they were incubated with the PAR-2 antibody (1:20) for 1 h at room temperature. Following incubation with the secondary antibody (1:300) conjugated with Alexa Fluor 488, the cells were washed and mounted in anti-fade medium (PermaFluor Mamtant Medium; Immunon, Pittsburgh, PA). The slides were examined under a fluorescence microscope (Leica, Cambridge, UK) at a 200x magnification using the appreciate filter.
Western blot and lectin blot analysis
The protein samples were electrophoresed on 12.5% polyacrylamide gels using Mini Protean II electrophoresis tanks (Bio-Rad, Hercules, CA). After electrophoresis, proteins were transferred to polyvinylidine difluoride (PVDF) membranes (Immobilon-P, 0.45 µm, Millipore, Bedford, MA,) at 240 mA for 60 min. Blots were blocked for 2 h with 5% skim milk in TBS-T (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) or with 5% BSA in TBS-T. Following incubation with the appropriate primary antibodies or fucose lectin overnight, the slides were washed. After washing, the blots were incubated with the corresponding secondary antibodies conjugated with HRP or ABC reagent (Vector Laboratories). Finally, specific proteins were visualized using an ECL system (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The fucose lectins used were AOL and AAL (Yazawa et al., 1984), both of which preferentially recognize core fucosylation on N-glycans.
Phosphorylation status of EGFR
Serum starvation of TGP49 cells was accomplished by substitution of 2% (w/v) BSA for 10% FBS in the culture medium before the experiments. Stimulations were carried out at 37°C in 25 mM Tris–HCl (pH 7.4) including 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, and 2% (w/v) BSA. Serum-starved cells were treated with EGF (1 nM) for 0, 5, 15, and 30 min. Stimulations were terminated by washing the cells once with ice-cold PBS supplemented with 0.4 mM sodium orthovanadate and then harvested by scraping. Cells were collected and solubilized for 15 min at 4°C in lysis buffer (50 mM Tris–HCl [pH 8.0], 1% [v/v] Triton X-100, 150 mM NaCl, 10% [v/v] glycerol, 2 mM EDTA, 100 µM phenylmethylsulfonylfluoride, 5 µg/mL of leupeptin, 1 of µg/mL aprotinin, 100 mM NaF, and 1 mM sodium orthovanadate). After lysis, the lysate was centrifuged at 20,000 g for 10 min at 4 °C to precipitate insoluble materials, and the detergent extracts (supernatant) were subjected to immunoprecipitation or directly electrophoresed and immunoblotted, as indicated below.
Immunoprecipitation
For immunoprecipitation, cell extracts (5001000 µg) were mixed with 20 µL of a 50% suspension of Protein G-Sepharose (Amersham Pharmacia Biotech AB) and incubated at 4°C for 2 h with continuous rotation. Ten microliters of polyclonal anti-EGFR Ab or anti-PAR-2 IgG was added and incubated at 4°C overnight with gentle agitation. The beads were washed four times with lysis buffer. The immunoprecipitated samples were eluted from the protein G-Sepharose by heating at 100°C for 5 min in Laemmli sample buffer with or without 2-mercaptoethanol. After centrifugation, the supernatants were resolved by SDS–PAGE and western blotted.
Statistical analysis
The results are expressed as mean value ± standard deviation (SD). Statistical analyses were carried out using Student’s t-test. A p-value <0.05 was considered statistically significant.
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/). Fig. 1:Real-time PCR analysis of various glycosyltransferases in TGP49 derived cells. Fig. 2:uPA expressions in cell culture media of TGP49 derived cells. Concentrated cell culture supernatants of TGP49-derivative cells were run on 10% gel and probed with a rabbit anti-uPA polyclonal antibody. CBB staining is shown as a loading control.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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We thank Dr Ihara of Osaka University for the GnGn-Asn-PABA acceptor. We also thank Dr Milton S. Feather for editing the manuscript. This work was supported by a CREST and the 21st Century COE Program from the Ministry of Education, Science, Culture, Sports, and Technology of Japan. Funding to pay the Open Access publication charges for this article was provided by the authors.
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Footnotes |
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1 These authors contributed equally to the work.
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Abbreviations |
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AAL, Aleuria aurantia lectin; AOL, Aspergillus oryzae lectin; BIM, bisindolylmaleimide; BSA, bovine serum albumin; dpc, days postcoitum; CCK-8, Cell Counting Kit-8; EDTA, ethylenediaminetetraacetic acid; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; Fut8,
1,6-fucosyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GnT III, N-acetylglucosaminyltransferase III; HRP, horseradish peroxidase; Ig, immunoglobulin; PAR-2, proteinase-activated receptor 2; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PKC, protein kinase C; PVDF, polyvinylidine difluoride; siRNA, short interfering RNA; RT–PCR, reverse transcription–polymerase chain reaction; TGF-ß, transforming growth factor-ß; TPA, 12-O-tetradecanoylphorbol-13-acetate; uPA, urokinase-type plasminogen activator; ß4GalT-I, ß1,4-galactosyltransferase I |
References |
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