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1 Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan
2 Department of Obstetrics and Gynecology, Graduate School of Medicine, Gunma University, 3-39-22 Showa-Machi, Maebashi, Gunma 371-8511, Japan
3 Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan
4 Pharmaceutical Development Laboratories, Kirin Brewery Co. Ltd., Takasaki, Gunma 370-1295, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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Stomach cancer-associated protein tyrosine phosphatase-1 (SAP-1) (also known as PTPRH) was originally identified as a PTP expressed in a human stomach cancer cell line (Matozaki et al. 1994). It is a receptor-type PTP (RPTP, R3 subtype) (Andersen et al. 2001), with a single catalytic domain in its cytoplasmic region and eight fibronectin type III-like domains in its extracellular region (Matozaki et al. 1994). Although its expression was found to be markedly increased in human colon and pancreatic cancers (Matozaki et al. 1994; Seo et al. 1997), the expression and localization of SAP-1 in normal tissues have not been well defined. A "substrate-trapping" approach identified p130Cas, a prominent focal adhesion-associated component of the integrin signaling pathway, as a likely physiological substrate of SAP-1 (Noguchi et al. 2001). Forced expression of SAP-1 in cultured cells also resulted in the dephosphorylation of several additional focal adhesion-associated proteins, including focal adhesion kinase and paxillin, as well as in impairment of reorganization of the actin-based cytoskeleton (Noguchi et al. 2001), suggesting that SAP-1 regulates the latter process. Forced expression of this PTP also inhibited cell proliferation, an effect that was mediated in part either by attenuation of growth factor-induced mitogen-activated protein kinase activation or by induction of caspase-dependent apoptosis (Noguchi et al. 2001; Takada et al. 2002). The cytoplasmic region of SAP-1 binds directly to Lck, and over-expression of SAP-1 resulted in down-regulation of the kinase activity of Lck and consequent inhibition of T cell receptor-mediated T cell function (Ito et al. 2003). However, given that most of these observations were made with cultured cells, the physiological or pathological functions of SAP-1 in vivo remain unknown.
We have now determined the sequence of mouse SAP-1 cDNA and generated a mAb to the mouse protein. With the use of these materials, we have shown that SAP-1 is expressed predominantly in the gastrointestinal tract, most prominently in the small intestine, and that it is specifically localized at the microvilli of gastrointestinal epithelial cells. Moreover, our observations suggest that SAP-1 plays an important role in the regulation of intestinal tumorigenesis.
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Results |
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To examine the expression of SAP-1 in various tissues, we first cloned a cDNA encoding mouse SAP-1. Searching of mouse Expressed Sequence Tag (EST) and other DNA data bases revealed several mouse DNA or EST sequences with similarity to those of human SAP-1 cDNA. With the use of these sequences, we obtained partial cDNA fragments for mouse SAP-1 by reverse transcription-polymerase chain reaction (RT-PCR) as described in Experimental procedures. We further searched the mouse cDNA data base provided by the Functional Annotation of the mouse (FANTOM) project (Carninci et al. 2005) for sequences similar to those of the partial mouse SAP-1 cDNA fragments and thereby obtained a full-length mouse SAP-1 cDNA (approximately 3.5 kb) (DDBJ Accession No. AB217856 [GenBank] ). The predicted structure of the mouse SAP-1 protein, which comprises 971 amino acids, was found to be similar to that of human SAP-1 (Fig. 1A), with the exception that the mouse protein contains six fibronectin type III-like domains in its extracellular region, whereas the human protein contains eight such domains (Matozaki et al. 1994). The amino acid sequences of mouse and human SAP-1 proteins share an overall identity of 56%, with the corresponding values for the extracellular and intracellular regions being 43% and 77%, respectively (Fig. 1A). A search of the Novartis GeneAtlas <http://symatlas.gnf.org/symatlas> with the mouse cDNA sequence revealed that SAP-1 mRNA is abundant in the small intestine and colon. Indeed, Northern blot analysis showed that mouse SAP-1 mRNA was preferentially distributed to the small intestine, stomach and colon (Fig. 1B).
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Specific localization of mouse SAP-1 to microvilli of gastrointestinal epithelial cells
We next examined the localization of SAP-1 in the small intestine. Immunohistofluorescence analysis with the mAb to SAP-1 revealed that SAP-1 was localized at the apical surface of intestinal epithelial cells (Fig. 2A), whereas β-catenin was present at sites of cell–cell adhesion as well as in the cytoplasm of epithelial cells, as described previously (Sena et al. 2006). The localization of SAP-1 was similar to that of ezrin or alkaline phosphatase (Fig. 2B), both of which are known to be localized at the microvilli of the intestinal epithelium (Narisawa et al. 2003; Saotome et al. 2004). Furthermore, the localization of SAP-1 overlapped with that of ezrin/radixin/moesin binding phosphoprotein of 50 kDa (EBP-50) at the apical surface of the intestinal epithelium (Fig. 2C). EBP-50 is a protein that links CD44 at the cell surface to ezrin and the actin cytoskeleton and is thus concentrated at the microvilli of the intestinal epithelium (Tsukita & Yonemura 1999). Each microvillus at the brush border of intestinal epithelial cells contains a bundle of actin filaments that extends the entire length of the microvillus as well as into the actin filament meshwork below it known as the terminal web (Mooseker 1985; Tyska et al. 2005; Ross & Pawlina 2006). SAP-1 immunoreactivity was detected immediately above the prominent staining of F-actin revealed by phalloidin, which may correspond to the terminal web, at the brush border of intestinal epithelial cells (Fig. 2C). Immuno-electron microscopy (EM) with the mAb to SAP-1 also revealed prominent staining at the microvilli of intestinal epithelial cells from wild-type mice (Fig. 2D), whereas such staining was virtually undetectable in sections from SAP-1-deficient mice, the generation of which is described below. These results thus indicated that SAP-1 is specifically expressed in intestinal epithelial cells and localizes to the microvilli of these cells.
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To investigate the physiological or pathological roles of SAP-1 in vivo, we generated SAP-1-deficient mice by targeted deletion of a fragment of genomic DNA containing exons 17 and 18 of the SAP-1 gene, which encode a region of the protein (amino acids 778–872) required for enzymatic activity. We generated a Sap1 mutant allele (Sap1flox) by introducing LoxP sites into the introns flanking exons 17 and 18 (Fig. 5A). Deletion of these two exons would be expected to introduce a frameshift mutation, resulting in loss of the COOH-terminal half of the catalytic domain as well as the downstream region of the protein. Crossing of Sap1flox/flox mice with CAG-Cre transgenic animals (Sakai & Miyazaki 1997) resulted in the generation of SAP-1-deficient (SAP-1 KO) mice. Southern blot analysis confirmed the presence of the deleted allele in SAP-1 KO mice (Fig. 5B). Immunoblot analysis with the mAb to SAP-1 also demonstrated the complete loss of SAP-1 protein or a reduction in its abundance of approximately 50% in the intestine of SAP-1 KO (Sap1–/–) mice and Sap1+/– heterozygotes, respectively (Fig. 5C). Immunohistofluorescence staining with this mAb also revealed the complete loss of SAP-1 in the apical brush border of the small intestine of SAP-1 KO mice (Fig. 5D). Given that the mAb to SAP-1 reacts with the extracellular region of the protein, we also performed immunoblot analysis with polyclonal antibodies to the cytoplasmic region of mouse SAP-1. This analysis also revealed the complete ablation of SAP-1 in the intestine of SAP-1 KO mice (data not shown). These results thus indicated that the entire SAP-1 protein is absent or undetectable in the intestine of SAP-1 KO mice, likely as a result of instability of the mutant SAP-1 mRNA or its protein product. We attempted to generate mice with microvillus-specific deficiency of SAP-1, but we found that the expression level of SAP-1 in Sap1flox/flox mice was reduced by > 50% relative to that in wild-type animals (data not shown). The SAP-1 KO mice were therefore backcrossed onto the C57BL/6 background for more than four generations, and offspring homozygous for the wild-type (WT) or mutant Sap1 alleles were studied in the following experiments.
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SAP-1 KO mice manifested normal morphology of intestinal epithelial cells and normal blood parameters. We next examined the possible effect of SAP-1 ablation on intestinal tumorigenesis, given that our previous studies with cultured cells suggested that SAP-1 regulates cell proliferation through its PTP activity (Noguchi et al. 2001; Takada et al. 2002). We analyzed intestinal tumorigenesis by crossing SAP-1 KO mice with mice of the Min/+ genotype for the adenomatous polyposis coli gene (Apc), a widely used model characterized by the spontaneous development of intestinal adenomas (Su et al. 1992; Rakoff-Nahoum & Medzhitov 2007). The numbers of visible adenomatous polyps (
1 mm in diameter) that formed in the small intestine and colon were quantified by stereoscopic microscopy. At 20 weeks of age, the total number of adenomas in the small intestine, but not that in the colon, appeared to be reduced in ApcMin/+Sap1–/– mice compared with that in ApcMin/+Sap1+/+ mice (Fig. 7A), although such reduction was not statistically significant. However, the number of macroadenomas ( 2 mm in diameter) in the intestine was markedly reduced in ApcMin/+Sap1–/– mice compared with that in ApcMin/+Sap1+/+ mice (Fig. 7B). These results thus suggested that SAP-1 regulates intestinal tumor growth in ApcMin/+ mice.
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Discussion |
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The predominant expression and localization of SAP-1 at the microvilli of gastrointestinal epithelial cells implicate SAP-1 in functions specific to these cells. The proper regulation of the actin cytoskeleton is important for maintenance of the morphology of intestinal microvilli (Fath & Burgess 1995). Indeed, mice that lack either ezrin or EBP-50, both of which are localized at microvilli and associated with the actin cytoskeleton, exhibit shortened and irregular microvilli in enterocytes (Morales et al. 2004; Saotome et al. 2004; Tamura et al. 2005). Moreover, protein tyrosine phosphorylation, which is determined by protein tyrosine kinases and PTPs, plays an important role in reorganization of the actin cytoskeleton (Giannone & Sheetz 2006). However, SAP-1-deficient mice manifested no marked changes in the morphology of intestinal epithelial cells, including that of microvilli or of tight or adherens junctions between these cells. Consistent with this finding, the expression of SAP-1 in the intestine was shown to be minimal during embryonic development and to increase markedly after birth, suggesting that SAP-1 is not important for determination of cell architecture in the intestinal epithelium.
Intestinal epithelial cells play an essential role in food digestion and in the absorption of nutrients and electrolytes through the intestine. Indeed, several digestive enzymes as well as channels and transporters for electrolytes or nutrients, including those for glucose, amino acids and fatty acids, are associated with the gastrointestinal microvilli (Holmes & Lobley 1989; Ross & Pawlina 2006). However, general nutrition status appeared unaffected in SAP-1 KO mice, as were peripheral blood cell counts. SAP-1 may thus be dispensable for regulation of food digestion and absorption of nutrients and electrolytes in the intestine. We are not able to exclude the possibility, however, that SAP-1 indeed contributes to such functions and that other PTPs, such as other RPTPs of the R3 subtype, are able to compensate for the loss of SAP-1 activity in SAP-1 KO mice.
We also found that SAP-1 deficiency inhibited tumorigenesis in mice heterozygous for mutation of Apc. The number of large adenomas ( 2 mm in diameter) in SAP-1 KO mice was markedly reduced whereas that of small adenomas (< 2 mm) was similar to that in WT mice, suggesting that SAP-1 contributes to tumor expansion but not to the initial transformation of normal epithelial cells into dysplastic cells. Functional impairment of the APC protein in ApcMin/+ mice is considered to result in stabilization and marked accumulation of β-catenin, which initiates transformation of normal epithelial cells and promotes tumorigenesis through constitutive activation of the β-catenin-transcription factor 4 (TCF-4) transcriptional pathway (Clarke 2006). Our results thus suggest that SAP-1 regulates intestinal tumorigenesis through cooperation with this pathway, although the precise mechanism of such regulation remains to be determined. Indeed, the rates of cell proliferation and apoptosis in adenomas of ApcMin/+Sap1–/– mice did not differ significantly from those apparent in ApcMin/+Sap1+/+ mice. In addition, the cytoplasmic and nuclear accumulation of β-catenin in adenomas were similar in mice of these two genotypes (unpublished data), suggesting that SAP-1 likely does not regulate the β-catenin-TCF-4 pathway itself. Instead, SAP-1 may promote intestinal cell proliferation through activation of protein tyrosine kinases such as Src family kinases. Indeed, PTP
, another RPTP, was shown to induce transformation of Rat-1 fibroblasts as a result of activation of Src mediated by dephosphorylation of its COOH-terminal tyrosine residue (Zheng et al. 1992). However, forced expression of SAP-1 failed to increase the activity of Src in cultured fibroblasts (unpublished data). Moreover, the levels of c-Src activity (in either adenomas or normal tissues) of ApcMin/+Sap1–/– mice did not differ from those of ApcMin/+Sap1+/+ mice (unpublished data). We previously showed that the expression of SAP-1 is markedly increased in human colon cancers (Matozaki et al. 1994; Seo et al. 1997). Our present results thus support the notion that SAP-1 promotes the tumorigenic potential of intestinal epithelial cells. By contrast, Src kinase may not be responsible for promotion by SAP-1 of intestinal tumorigenesis. Recent studies also suggests the transforming properties of PTPs. Gain of function mutations in SHP-2 are implicated to cause juvenile myelomonocytic leukemia (Tartaglia et al. 2003). In addition, deletion of PTP1B activity delays mammary tumorigenesis and protects lung metastasis in transgenic mice with an active mutant of ErbB2 (Julien et al. 2007). Further studies are warranted to characterize the physiological and pathological roles of SAP-1 as an intestinal epithelium-specific PTP.
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Experimental procedures |
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Mouse mAbs to β-tubulin (TUB 2.1) or to actin (AC-40) were obtained from Sigma (St. Louis, MO), those to β-catenin or to p120-catenin were from BD Biosciences (San Diego, CA), and that to E-cadherin (ECCD-2) was from Takara BIO (Otsu, Shiga, Japan). Rabbit polyclonal antibodies to EBP-50 were from Abcam (Cambridge, UK) and those to claudin-15 were kindly provided by M. Furuse (Kobe University, Kobe, Japan). A rat mAb to CD31 (MEC13.3) was from BD Biosciences and that to ezrin (M11) was kindly provided by S. Tsukita (Osaka University, Suita, Japan). Rabbit polyclonal antibodies to ZO-1, rhodamine-conjugated phalloidin, and secondary antibodies labeled with fluorescein isothiocyanate (FITC), Cy3 or Alexa488 for immunofluorescence analysis were obtained from Invitrogen (Carlsbad, CA). Horse radish peroxidase (HRP)-conjugated goat polyclonal antibodies to rat, mouse or rabbit IgG for immunoblot analysis were from Jackson ImmunoResearch (West Grove, PA). FITC-conjugated UEA-I was from Vector (Burlingame, CA).
Isolation of a full-length mouse SAP-1 cDNA
To obtain a full-length mouse SAP-1 cDNA, we searched mouse data bases for sequences similar to that of human SAP-1 cDNA with the use of the Basic Local Alignment Search Tool (BLAST) program (NCBI). Several EST fragments as well as genomic DNA fragments potentially corresponding to the mouse SAP-1 gene were identified and were used to obtain partial cDNAs for mouse SAP-1 by RT-PCR. The sequences of two RT-PCR products (226 and 431 bp) were then used to search the mouse DNA data base provided by the FANTOM project (Carninci et al. 2005), resulting in the identification of a cDNA clone (#9030616J04) that contained the full-length SAP-1 cDNA.
Generation of monoclonal and polyclonal antibodies to mouse SAP-1
A rat mAb to mouse SAP-1 (clone 123) was generated with the use of a recombinant fusion protein comprising the extracellular region of mouse SAP-1 (amino acids 1–604) and the Fc region of human IgG as the antigen. Rabbit polyclonal antibodies to mouse SAP-1 were also generated with the use of a glutathione-S-transferase fusion protein containing the cytoplasmic region of mouse SAP-1 (amino acids 648–971) as antigen, as previously described (Matozaki et al. 1994).
Northern blot analysis
A digoxigenin-labeled cRNA probe corresponding to nt –31 to 3454 of mouse SAP-1 cDNA was synthesized with the use of a DIG RNA Labeling Kit (Roche, Basel, Switzerland) and T7 RNA polymerase. Polyadenylated RNA (2 µg) fixed on a nylon membrane was subjected to hybridization overnight at 50 °C with 400 ng of the cRNA probe in DIG Easy Hyb (Roche). The membrane was then exposed to alkaline phosphatase–conjugated sheep polyclonal Fab fragments to digoxigenin (1 : 10 000 dilution; Roche), and hybridization complexes were visualized with the CDP-Star reagent (Roche).
Immunoblot analysis
Mouse tissues were homogenized on ice in lysis buffer (20 mM Tris–HCl (pH 7.6), 140 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 µg/mL), and 1 mM sodium vanadate. The lysates were centrifuged at 10 000 g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoblot analysis.
Histological and immunofluorescence analyses
For analysis of mouse tissues, mice were anesthetized by intraperitoneal (i.p.) injection of sodium pentobarbital at 25 mg/kg of weight and were then perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Tissues were then dissected, fixed again with 4% PFA in PBS for 1 h at room temperature, transferred to a series of sucrose solutions [7%, 20% and 30% (w/v), sequentially] in PBS, embedded in optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan), and rapidly frozen in liquid nitrogen. Frozen sections of 5 µm thickness were then prepared and subjected to immunofluorescence analysis with primary antibodies and secondary antibodies labeled with fluorescent dyes, as described previously (Ishikawa-Sekigami et al. 2006). Fluorescence images were obtained with a confocal laser-scanning microscope (LSM Pascal; Carl Zeiss, Jena, Germany). To examine the expression of alkaline phosphatase, we used a Vector Red Alkaline Phosphatase Kit I (Vector Laboratories, Burlingame, CA). Fixed tissue samples were also embedded in paraffin, sectioned, and stained with Mayer's hematoxylin–eosin.
Transmission EM
Mice were anesthetized as described above and were then perfused transcardially with 2% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were removed, immersed in the same fixative for 1 h at 4 °C, and then incubated for 1 h at 4 °C with 1% OsO4 in the same buffer. They were then dehydrated and embedded in Epon. For immuno-EM, tissue samples were fixed by immersion for 2 h at room temperature in PBS containing 4% PFA and 1% glutaraldehyde. Frozen sections of 10 µm thickness were then prepared and processed for immunostaining with a mAb to mouse SAP-1, and with Fab fragments of goat antibodies to rat IgG that were labeled with 1.4-nm gold particles (Nanoprobes, Yaphank, NY); signals were enhanced with the use of an HQ Silver Enhancement kit (Nanoprobes). The sections were fixed again with 1% OsO4 and 0.1% potassium ferrocyanide and were finally embedded in Epon. Ultrathin sections (90 nm) were prepared, stained with uranyl acetate and lead citrate, and examined with a JEM 1010 electron microscope (JEOL, Tokyo, Japan).
Generation of SAP-1-deficient mice
To generate SAP-1-deficient mice, we first obtained two bacterial artificial chromosome clones containing 129/Sv mouse Sap1 genomic DNA from BACPAC Resources Center (Children's Hospital Oakland Research Institute, Oakland, CA). The targeting vector (Fig. 5A) was constructed with a 1.2-kb DNA fragment spanning exon 14 to intron 16 as a 5' homologous region, with a 6.4-kb fragment spanning intron 16 to intron 21 as a 3' homologous region, with a construct (PGKβgeo) comprising the promoter of the mouse phosphoglycerate kinase 1 gene linked to a β-galactosidase–neomycin phosphotransferase fusion gene (Friedrich & Soriano 1991), and with a diphtheria toxin A chain (DTA) gene cassette (Takara BIO). A LoxP site was also inserted into intron 16 as well as into intron 18 of the targeting vector. The targeting vector was introduced into the J1 line of mouse embryonic stem cells by electroporation, and homologous recombination was confirmed by Southern blot analysis, as described previously (Harada et al. 1994), with two DNA probes (S1 and S2) (Fig. 5A). Chromosomal DNA was thus purified from each cell clone and was digested with Hind III (for the S1 probe) or Bam HI (for the S2 probe). We crossed transgenic mice that express Cre recombinase under the control of CAG promoter (CAG-Cre mice) (Sakai & Miyazaki 1997) with mice harboring a targeted Sap1 allele. The genotype of the resulting offspring was determined by either Southern blot (Fig. 5B) or PCR analysis. In PCR analysis, 396- and 496-bp DNA fragments corresponding to the wild-type and targeted alleles, respectively, were generated with the sense primer MSAP-MS (5'-ATGGAGAGACT GGCTCTTGC-3') and the antisense primer MSAP LOX-AS (5'-CCATGCAGCTAGTAACATGG-3') and a 300-bp DNA fragment corresponding to the deleted allele was generated with the primers Lox-g (5'-ACGGTATCGATAAGCTTGGC-3') and MSAP LOX-AS. SAP-1-KO mice were then backcrossed onto the C57BL/6 background for four generations. Mice were bred and maintained at the Institute of Experimental Animal Research of Gunma University under specific pathogen-free conditions. All animal experiments were performed with the guidelines of the Animal Care and Experimentation Committee of Gunma University.
Adenoma formation in SAP-1-deficient mice with the ApcMin allele
ApcMin/+ mice (C57BL/6J-ApcMin/J) were obtained from the Jackson Laboratory (Bar Harbor, ME) and were crossed with SAP-1 KO mice. The resulting ApcMin/+Sap1+/– offspring were crossed to generate ApcMin/+Sap1–/– mice and their ApcMin/+Sap1+/+ littermates for comparison. The small intestine and colon were removed from mice at 20 weeks of age and were fixed in methacarn (methanol : chloroform : acetic acid, 4 : 2 : 1, v/v) for 3 min (Prokhortchouk et al. 2006). The number and diameter of adenomas were determined with the use of a stereoscopic microscope (SZX7; Olympus, Tokyo, Japan) at a magnification of 10x. Tissue was also embedded in paraffin for histological analysis.
BrdU incorporation assay
Mice were injected i.p. with BrdU (50 µg/g of body weight in PBS) and killed 1 h later. Intestinal tissue was fixed in methacarn, embedded in paraffin, sectioned, and stained with a mAb to BrdU with the use of a BrdU In-Situ Detection Kit (BD Pharmingen, Franklin Lakes, NJ). The BrdU labeling index was defined as the percentage of BrdU-positive cells per 150 adenoma cells.
Apoptosis assay
Intestinal tissue was fixed in methacarn, transferred to a series of sucrose solutions [7%, 20% and 30% (w/v), sequentially], embedded in OCT compound, and evaluated for apoptosis with an apoptosis detection kit (Takara BIO). The number of apoptotic cells per 150 adenoma cells was determined.
Statistical analysis
Data are presented as means ± SEM and were analyzed by Student's t test. A P value of < 0.05 was considered statistically significant.
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Acknowledgements |
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Footnotes |
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These authors have contributed equally to this work. 
* Correspondence: matozaki{at}showa.gunma-u.ac.jp |
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Received: 5 November 2008
Accepted: 21 November 2008
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