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Originally published as JHC exPRESS on June 13, 2005.
doi:10.1369/jhc.4A6508.2005
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Volume 53 (11): 1371-1382, 2005
Copyright ©The Histochemical Society, Inc.
The Subcellular Localization of Syntaxin 17 Varies Among Different Cell Types and Is Altered in Some Malignant Cells
Departments of Gastrointestinal Medical Oncology (QZ,JL,JLA,LH) and Pathology (MD), University of Texas M. D. Anderson Cancer Center, Houston, Texas
Correspondence to: Linus Ho, Department of Gastrointestinal Medical Oncology, 1515 Holcombe Blvd., #426, Houston, TX 77030. E-mail: linusho{at}mdanderson.org
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Summary |
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Syntaxin 17 (STX17) is a divergent member of the syntaxin family of proteins first discovered by Scheller and colleagues in a yeast two-hybrid screen designed to identify novel mammalian SNAREs (soluble N-ethylmaleimide–sensitive factor-attachment protein receptors). We recently independently identified STX17 as a novel Ras-interacting protein, but immunohistochemical studies suggested that STX17 is localized to the nucleus in normal pancreatic ductal epithelial, acinar, and islet cells in contrast to previous reports of cytoplasmic localization, albeit in other cell types. Therefore, we have conducted a more thorough survey of various human and mouse tissues to better establish the expression pattern of STX17 in different tissues and cell types. Although RT-PCR experiments demonstrate ubiquitous expression of STX17, closer examination by immunohistochemistry reveal that STX17 expression is limited to certain cell types. Furthermore, in contrast to the cytoplasmic localization previously reported in a limited number of cell types, we find that in many other cell types, syntaxin 17 can be found in the nucleus. Finally, we demonstrate that in human hepatocellular carcinoma cell lines, STX17 localization is altered relative to normal hepatocytes, although the localization of STX17 differs even between these established human cancer cell lines and fresh human hepatocellular carcinoma cells, emphasizing the caution that must be exercised in drawing conclusions from data gathered in cell lines. The sequence divergence of STX17, the unexpected nuclear localization of STX17 in many cell types, and the altered localization of STX17 in malignant cells argue for a novel function of syntaxin 17 distinct from its hypothesized role in mediating membrane fusion events. (J Histochem Cytochem 53:1371–1382, 2005)
Key Words: syntaxin 17 • subcellular localization • hepatocellular carcinoma
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Introduction |
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THE SYNTAXINS COMPRISE A FAMILY of proteins that is principally involved in mediating membrane fusion events critical in regulating vesicular transport among various intracellular membrane compartments. Under the SNARE (soluble N-ethylmaleimide–sensitive factor-attachment protein receptor) hypothesis proposed by Rothman and colleagues, docking and fusion between donor and acceptor membranes are mediated by interaction among compartment-specific SNARE proteins located within the donor (vesicular) and acceptor (target) membranes, generically called v-SNAREs and t-SNAREs, respectively. v-SNAREs are represented by members of the vesicle-associated membrane proteins family, and t-SNAREs are represented by members of the syntaxin and soluble NSF attachment protein families.
In general, all mammalian syntaxins are type II transmembrane proteins with a cytoplasmic N terminus and a single C-terminal transmembrane domain (syntaxin 11 is exceptional in that it lacks a transmembrane domain, although it is nonetheless predominantly membrane associated). However, all syntaxins share a conserved, membrane-proximal, 
-helical, coiled-coil SNARE domain that mediates the interaction between SNAREs, leading to formation of a parallel -helical bundle comprising four chains, called the SNARE complex. In 1998, Steegmaier and colleagues reported the early characterization of two novel syntaxins, including syntaxin 17 (STX17), which was discovered in a two-hybrid screen using syntaxin 3 as the bait (Steegmaier et al. 1998
). Subsequent analysis of STX17's peptide sequence and comparison with other syntaxin sequences showed that STX17 is the most divergent known member of the syntaxin family (Teng et al. 2001), although it does retain homology in the conserved membrane-proximal coiled-coil domain that defines the syntaxin family (Steegmaier et al. 1998). Steegmaier et al. later reported that STX17 expression is particularly high in "steroidogenic cells" located in the adrenal gland, liver, ovary, placenta, and testis, where STX17 appears to be localized to the smooth endoplasmic reticulum (ER) (Steegmaier et al. 2000). On the basis of these and other data linking STX17 to proteins such as rbet1, rsec22b, and rsly1, they hypothesized that STX17 functions in vesicle trafficking to the smooth ER.
Our primary interest has been in understanding pancreatic tumorigenesis, and considerable evidence suggests that K-Ras plays a critical role in this process. For example, the vast majority (at least 80%) of pancreatic adenocarcinomas express mutated and constitutively activated K-Ras, inhibition of Ras activity through a variety of means leads to abrogation of the malignant phenotype in pancreatic cancer cells (Kita et al. 1999; Gana-Weisz et al. 2002), and conditional overexpression of mutant K-Ras in pancreatic progenitor cells leads to preneoplastic lesions (Hingorani et al. 2003). Therefore, we performed a yeast two-hybrid screen to identify novel Ras-interacting proteins, one of which was found to be STX17 (unpublished data). However, when we performed immunohistochemical studies to determine the pattern of expression of STX17 in pancreatic tissue, we were surprised to find that STX17 is, in fact, expressed in cells that would not be considered "steroidogenic" and, in many cases, exhibits nuclear localization. As a result, we have explored more comprehensively the expression pattern of STX17 in many mouse and human tissues, and we report that, although STX17 is ubiquitously expressed at the tissue level, its expression is highly cell-specific. Furthermore, in different cell types, STX17 can be localized to the cytoplasm, nucleus, or both, and its subcellular localization may be altered in malignant cells compared with their normal counterparts. The highly divergent sequence of STX17, the unexpected localization of STX17 in the nuclei of nonsteroidogenic cells, and its altered localization in malignant cells all suggest a novel function for STX17 distinct from its hypothesized role in vesicle transport between the ER and Golgi.
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Materials and Methods |
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Generation of a Rabbit Polyclonal Antibody
A 15-residue peptide corresponding to the N-terminal 15 amino acids of human STX17 (MSEDEEKVKLRRLEP) was synthesized and used to generate a rabbit polyclonal antibody, which was subsequently affinity purified (all performed by Sigma-Genosys; The Woodlands, TX).
Cloning
Full-length human STX17 was PCR-cloned into pCR-Blunt II-TOPO (Invitrogen; Carlsbad, CA) using the primers described below and sequenced to confirm its veracity. The full-length hSTX17 coding sequence was then released using the flanking BamHI and XhoI sites and cloned into pCMV-Tag1 (Stratagene; La Jolla, CA) linearized with Bgl II and XhoI, resulting in hSTX17 with an N-terminal FLAG tag. This FLAG-tagged version of hSTX17 was then, in turn, released with BamHI and XhoI and cloned into pcDNA3.1(+)/myc-His A (Invitrogen) linearized with BamHI and XhoI, finally resulting in full-length hSTX17 with an N-terminal FLAG tag and C-terminal Myc and (His)6 tags.
RT-PCR
Aliquots of total RNA isolated from various human tissues were purchased from Clontech (subsidiary of BD Biosciences; Mountain View, CA) and used to generate single-stranded cDNA using the Advantage for RT-for-PCR kit (Clontech) according to the manufacturer's instructions. PCR was performed using Pfu Turbo DNA polymerase (Stratagene) and the following glyceraldehyde phosphate dehydrogenase (GAPDH) and STX17-specific primers: forward primer for GAPDH (5'-CAAGATCATCAGCAATGCCTCCTG-3'), reverse primer for GAPDH (5'-CCTGCTTCACCACCTTCTTGATGTC-3'), forward primer for STX17 (5'-TCCATGACTGTTGGTGGAGCA-3'), and reverse primer for STX17 (5'-CAGCTGCAATTCCTGCCACTT-3'). The PCR reactions were performed using the following settings on an MJ Research (subsidiary of BioRad; Hercules, CA) PTC-100 Peltier Thermal Cycler: 3 min at 94C (preheating); cycles of 45 sec at 94C, 2 min at 58C, and 5 min at 72C (25 cycles for GAPDH and 40 cycles for STX17); and 10 min at 72C (final extension). The resultant PCR products were electrophoresed in 2.0% agarose at 125V.
Tissue Culture
AsPC-1, BxPC-3, Capan-1, HEK 293, HepG2, MiaPaCa-2, PLC/PRF/5, and SNU-449 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Capan-1, HEK 293, and MiaPaca-2 cells were grown in DMEM; AsPC-1, BxPC-3, and SNU-449 cells were grown in RPMI 1640; and HepG2 and PLC/PRF/5 cells were grown in Eagle's minimal essential medium, all supplemented with 10% FBS, 100 U/ml penicillin, and 100 mcg/ml streptomycin and grown at 37C in a humidified atmosphere of 5% CO2.
Transfection
HEK 293 cells were transiently transfected in 6-well plates with 1 µg DNA and 3 µl FuGENE 6 (Roche Applied Science; Indianapolis, IN) according to the manufacturer's instructions. Cells were then harvested for Western blot analysis after 
72 hr.
Immunohistochemistry
Five-µm sections of formalin-fixed, paraffin-embedded tissue samples were prepared and placed onto glass slides. The samples were deparaffinized with Histoclear and rehydrated with serial alcohol washes. After microwave-based antigen retrieval, the sections were incubated with the primary antibody overnight at 4C. Bound antibody was detected using the corresponding biotinylated secondary antibody, ABC kit, and DAB kit (Vector Labs; Burlingame, CA). Hematoxylin was used for counterstaining. Imaging was performed using a Zeiss Axioskop microscope (Zeiss; Thornwood, NY).
For immunohistochemical studies on human tumor cell lines, cells were plated onto 4- or 8-well chamber slides and grown for 24–48 hr before analysis. The cells were then washed in PBS, fixed for 20 min in 4% paraformaldehyde, and permeabilized in 0.2% Triton X-100. The cells were sequentially blocked with 10% horse serum and incubated overnight with the appropriate primary antibody at 4C. Bound antibody was detected using the corresponding biotinylated secondary antibody, ABC kit, and DAB kit (Vector Labs). Hematoxylin was used for counterstaining. Imaging was performed using a Zeiss Axioskop microscope.
Immunofluorescence
Slides bearing 5-µm sections of formalin-fixed, paraffin-embedded tissue samples were deparaffinized with Histoclear and rehydrated with serial aqueous alcohol incubations. After microwave-based antigen retrieval, the sections were incubated with anti-STX17 polyclonal antibody at 4C for 12 hr. Bound antibody was detected with fluorescent dye-labeled secondary antibody (Alexa Fluor 488-conjugated anti-rabbit IgG; Molecular Probes, Eugene, OR) at a dilution of 1:1000 and imaged using an Olympus Fluoview FV500 laser-scanning microscope (Olympus; Melville, NY).
Mouse Samples
Slides with archival formalin-fixed and paraffin-embedded samples from various representative mouse tissues were kindly provided by Dr. Carolyn van Pelt (Department of Veterinary Medicine and Surgery, M. D. Anderson Cancer Center).
Patient Samples
Formalin-fixed tissues remaining after pathologic examination of surgical samples were paraffin-embedded, cut into 5-µm sections, and placed onto slides for further immunohistochemical analysis. Use of residual human tissue was approved by the Institutional Review Board of M. D. Anderson Cancer Center.
Cell Fractionation Experiments
Cytoplasmic and nuclear cell extracts were prepared using a NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology; Rockford, IL) according to the manufacturer's instructions. Extracts were then electrophoresed on 12% SDS-PAGE gels, transferred to Immobilon-P membrane (Millipore; Billerica, MA), and hybridized with the affinity-purified rabbit polyclonal antibody against STX17 (1:1000 dilution of a stock 0.2 mg/ml solution) or with a mouse anti-PARP [poly(ADP-ribose) polymerase] antibody [1:1000 dilution, clone #7D3-6; BD Transduction Laboratories (BD Biosciences)] for 2 hr at room temperature. The membrane was developed using ECL Western blotting detection reagents (Amersham Biosciences; Piscataway, NY) according to the manufacturer's instructions and exposed to X-ray film (Kodak; New Haven, CT).
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STX17 Demonstrates Cell Type–specific Expression and Subcellular Localization Patterns in Normal Human Pancreatic Tissue
Because K-Ras is commonly mutated and thereby constitutively activated in
80–90% of pancreatic ductal adenocarcinomas, we wanted to determine the expression pattern of STX17 in the normal pancreas as a prelude to further studies comparing normal and tumor tissue. Therefore, we generated a rabbit polyclonal antibody directed against the N-terminal 15 amino acids of STX17 and used it to stain normal mouse pancreatic tissue by immunohistochemistry and immunofluorescence. Surprisingly, these studies showed that STX17 is expressed quite strongly both in the cytoplasm and in the nuclei of ductal epithelial and islet cells (Figure 1A). On the other hand, levels of STX17 expression are significantly lower in acinar cells, the predominant cell type in the pancreas, and is predominantly cytoplasmic in localization, and stromal cells in the pancreas are consistently negative for STX17 expression. We have also performed immunofluorescence microscopy, which confirms these findings (data not shown).
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STX17 Expressed in the Cytoplasm of Steroidogenic Cells and Hepatocytes
In apparent contrast to our results, Steegmaier et al. have reported that STX17 is localized to the cytoplasm and, more specifically, to the smooth endoplasmic reticulum in hepatocytes and steroidogenic cells in the adrenal cortex and testis (Steegmaier et al. 2000
). To confirm their results and to determine whether our disparate results reflect nonspecific binding by our antibody or simply different patterns of expression in different cell types, we stained sections of adrenal gland, liver, and testis from both mouse and humans using our anti-STX17 antibody (Figure 1). In a low-magnification view of the murine adrenal gland, one can see that STX17 expression is primarily confined to cells in the zona fasciculata and zona reticulosa, sparing cells located in the zona glomerulosa and in the medulla (Figure 1B); a higher magnification view shows that STX17 is distributed diffusely throughout the cytoplasm in these cells (Figure 1C). This distinction is functionally significant because cells in each zone synthesize and secrete different products; specifically, the zona fasciculata and the zona reticulosa produce glucocorticoids and sex steroids, respectively, and the zona glomerulosa and the medulla produce mineralocorticoids and catecholamines, respectively. In murine liver, STX17 is expressed primarily in the cytoplasm of hepatocytes (Figure 1D), akin to but at higher levels than in pancreatic acinar cells (see previous); stromal cells (lower right-hand corner) are again negative for STX17 expression. In the human testis (Figure 2A), STX17 expression is seen in several different cell types, including Sertoli cells and germ cells at various stages of maturation, both of which are found within the seminiferous tubules, and Leydig cells, which are found in the interstitium; however, other cell types found in the interstitium do not express STX17. Thus, although we do confirm the cytoplasmic expression of STX17 in hepatocytes and certain steroidogenic cells in the adrenal gland, we find that STX17 is not necessarily expressed in all steroidogenic cells (e.g., zona glomerulosa cells of the adrenal gland), but can be detected at relatively high levels in testicular germ cells, which were not previously reported as being STX17-expressing.
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Confirmation of the Specificity of the Polyclonal Antibody
The lack of significant background staining and the consistent failure of specific cell types to stain for STX17 (e.g., stromal cells in the pancreas and testis) both suggest that our antibody is specific for STX17. However, we also performed blocking experiments using a nonspecific 15-mer peptide (Figure 2A) and a STX17-specific peptide (the 15-mer peptide used to generate the antibody; Figure 2B) in immunohistochemical studies. As shown, prior incubation with the STX17-specific 15-mer peptide totally blocks binding of our antibody, but prior incubation with an unrelated 15-mer peptide does not affect antibody binding.
To further demonstrate the specificity of our antibody, we constructed a vector expressing full-length human STX17 with a C-terminal Myc tag. We then compared the expression of endogenous and transfected STX17 in HEK 293 cells transfected with empty vector or vector expressing Myc-tagged STX17 (Figure 2C). In Lanes 1 and 2, Western blot analysis using a monoclonal antibody against the Myc tag demonstrates expression of Myc-tagged STX17 only in 293 cells transfected with the appropriate vector. In Lanes 3 and 4, we used our polyclonal antibody against STX17 to probe the same lysates, thereby demonstrating that our antibody specifically detects the same band as the monoclonal anti-Myc tag antibody. The lower bands seen in both Lanes 3 and 4 are felt to represent endogenous, untagged STX17, which would be expected to be present in both cell lysates. Finally, in Lanes 5 and 6, we preincubated the filter with a STX17-specific 15-mer peptide before probing with our anti-STX17 polyclonal antibody. The complete absence of antibody binding, including absence of the lower bands seen in Lanes 5 and 6, supports our conclusion that the polyclonal anti-STX17 antibody specifically detects human STX17 and that the lower molecular weight bands seen in Lanes 3 and 4 do, in fact, represent endogenous STX17.
STX17 Ubiquitously Expressed in Human Tissues
As a first approximation, we examined STX17 expression in a wide variety of human tissues by RT-PCR (Figure 3). These experiments demonstrate that STX17 is ubiquitously expressed in human tissues, as previously suggested by Northern blot analyses (Steegmaier et al. 1998,2000). However, as illustrated in the following section, ubiquitous expression at the tissue level does not necessarily translate into ubiquitous expression at the cellular level. A second finding is the consistent presence of two distinct STX17-specific PCR products. This suggests the presence of distinct STX17 isoforms, presumably resulting from alternative splicing.
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STX17 Expression in Other Tissues
Because RT-PCR analysis of gene expression in tissues yields only an aggregate view of the various cell types found within a given tissue type, one cannot discern whether or not STX17 expression might be limited to certain cell types within each tissue, and our data suggest that STX17 expression is indeed limited and specific to certain cell types. Therefore, we set out to more definitively catalog the expression pattern of STX17 by examining a extensive battery of normal mouse and human tissues by immunohistochemistry.
Gastrointestinal Tract
In the murine gastrointestinal tract, STX17 expression is quite variable. In the proximal esophagus, for example (Figure 4A), only the superficial squamous epithelial cells demonstrate intense cytoplasmic and nuclear staining with the underlying mucosa and submucosa largely devoid of STX17 expression; in contrast, skeletal muscle cells exhibit diffuse but exclusively cytoplasmic expression of STX17. In the stomach (Figure 4B), STX17 expression is most prominent at the base of the gastric pits with both chief and parietal cells demonstrating strong nuclear staining with lesser expression in the cytoplasm. Furthermore, there is a gradient of STX17 expression within the mucosal layer with the highest levels of expression seen at the base of the gastric pits and waning expression as one proceeds superficially toward the mucosal surface. In contrast to the skeletal muscle seen in the proximal esophagus, the underlying gastric smooth muscle displays both nuclear and cytoplasmic staining that seemingly varies from cell to cell. In the small intestine (Figure 4C), both nuclear and cytoplasmic localization of STX17 expression is seen in many cells, particularly the apical epithelial cells of the intestinal villi and some cells within the crypts. In the large intestine (Figure 4D), a somewhat similar pattern is seen with apical epithelial cells and basilar crypt cells both staining prominently for STX17. However, it is interesting to note that within the apical epithelial cells, STX17 is located primarily within the cytoplasm, whereas the crypt cells demonstrate both nuclear and cytoplasmic staining.
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Nervous System
Syntaxins 1A and 1B, the first members of the syntaxin family to be identified, have been localized to the plasma membrane and secretory granules of neurons and are involved in mediating neurotransmitter release. Therefore, we were interested in comparing the expression pattern of STX17 with that of more classical syntaxins in neural tissue. As shown in Figure 5, STX17 is strongly and expressed primarily in the nuclei of neurons in the central nervous system in stark contrast to the localization of syntaxins 1A and 1B. The most notable exceptions are the large Purkinje cells of the cerebellum (Figure 5B), which demonstrate both nuclear and perikaryal staining for STX17. However, there is no obvious STX17 expression either in the dendritic and axonal processes of neurons or in supporting cells such as the Schwann cells of peripheral nerves (the visualized nuclei belong to Schwann cells; Figure 5C).
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Genitourinary Tract
In the kidney (Figure 6A), most of the visualized epithelial cells comprising the various renal ducts and tubules express STX17 in a diffuse cytoplasmic pattern with less intense nuclear staining although some ductal cells seen in the upper and midportions of the figure demonstrate particularly strong nuclear staining (see arrows). On the other hand, as seen previously in many other tissues, most of the cells present in the interstitium lack significant STX17 expression. In the mouse bladder (Figure 6B) and ureter (data not shown), STX17 is highly expressed in the cytoplasm and nuclei of urothelial cells lining the genitourinary tract with relative sparing of the underlying submucosal layers in a pattern very similar to that seen earlier in the proximal esophagus (Figure 4A); in the bladder, the deeper smooth muscle cells exhibit cytoplasmic but not nuclear expression of STX17. In the human prostate, STX17 is only weakly expressed in the cytoplasm of ductal cells (broad arrows in Figure 6C); the strongly staining cells in the middle of the figure (thin arrows) are inflammatory cells present within a capillary. In the mouse endometrium (Figure 6D), there is relatively minimal STX17 expression except for a subset of glandular cells demonstrating weak cytoplasmic staining (arrows). In the Fallopian tube (Figure 6E), STX17 expression is seen predominantly in the cytoplasm of the columnar epithelial cells lining the tube itself. In the vagina (Figure 6F), as in the esophagus, the lining squamous cells stain strongly for STX17, both in the nucleus and in the cytoplasm, although in this case, many of the submucosal cell generally devoid of STX17 expression although some scattered cells in the stroma demonstrate weak to moderate expression of STX17.
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Muscle
The pattern of STX17 expression in both smooth and skeletal muscle is well demonstrated in the context of several tissues presented in this report. In smooth muscle (in the stomach in Figure 4B, in the bladder in Figure 6B, and in pulmonary bronchioles in Figure 7C), STX17 expression is predominantly cytoplasmic, although occasional nuclear localization is seen in the smooth muscle cells of the stomach. On the other hand, in skeletal muscle cells (for example, in the esophagus in Figure 2A and skin in Figure 7E), STX17 is exclusively localized to the cytoplasm. The expression pattern of STX17 in cardiac muscle cells (Figure 7A) is more akin to that in smooth muscle cells in that strong cytoplasmic expression predominates with occasional expression of STX17 in scattered nuclei.
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Other Miscellaneous Tissues
In mouse bone and cartilage (Figure 7B), both chondrocytes and osteoblasts exhibit relatively strong STX17 expression in both the nucleus and cytoplasm; interestingly, red blood cells also stain strongly for STX17. In the lungs (Figure 7C), the alveolar epithelial cells lack significant STX17 expression, whereas only smooth muscle cells comprising the bronchiole exhibit significant STX17 expression. In the thyroid gland (Figure 7D), follicular cells are devoid of STX17 expression. In a section of thin skin from the mouse (Figure 7E), one again sees very strong STX17 expression in squamous epithelial cells and in hair follicles, which are derived from the epidermal epithelium; however, higher magnification views demonstrate that most STX17 expression is localized to the cytoplasm in these cells (data not shown). As seen previously, skeletal muscle demonstrates cytoplasmic STX17 localization, as do cells comprising sebaceous glands and many of the cells in the dermis (between the adipose layer and the epidermis). Finally, in the human breast (Figure 7F), one sees that the acinar epithelial cells, which represent the majority of cells comprising the lobules, demonstrate predominantly cytoplasmic localization of STX17 whereas myoepithelial cells (arrow) exhibit both nuclear and cytoplasmic expression of STX17 and the stroma exhibits minimal STX17 expression.
Syntaxin 17 Localization Altered in Some Tumor Cells Relative to Normal Cells
To compare the subcellular localization of STX17 in malignant cells with their normal counterparts, we examined the localization of STX17 in several human pancreatic ductal adenocarcinoma (AsPC-1, BxPC-3, Capan-1, and MiaPaca-2) and hepatocellular carcinoma (HepG2, PLC/PRF/5, and SNU-449) cell lines by both immunohistochemistry and immunofluorescence. These cell lines were chosen because of the distinct patterns of STX17 localization in normal pancreatic ductal epithelial cells and hepatocytes. In concert with normal pancreatic ductal epithelial cells, all four of the sampled pancreatic cancer cell lines exhibit strong, predominantly nuclear expression of STX17 with lesser perinuclear cytoplasmic staining (a representative microphotograph of AsPC-1 cells is shown in Figure 8A). However, in contrast to normal hepatocytes, all three hepatocellular cell lines demonstrate nuclear localization of STX17 (a representative microphotograph of HepG2 cells is shown in Figure 8B).
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We then examined the pattern of STX17 expression in archival tissue obtained from six patients who underwent resection for hepatocellular carcinoma. Somewhat surprisingly, we found that in this random sample of patients, STX17 is primarily localized in the cytoplasm in five of these six specimens (e.g., Figure 8D). Interestingly, the specimen displaying nuclear localization (Figure 8C) came from the only patient with hepatitis B. These results serve as a cautionary note in extrapolating from cell lines to fresh tumors in vivo and also raise interesting questions about the mechanism of intracellular STX17 localization.
Cell Fractionation Experiments Confirm the Cytoplasmic and Nuclear Localization of STX17 in Normal and Malignant Hepatocytes, Respectively
To independently confirm the differential localization of STX17 in normal and malignant hepatocytes, we isolated cytoplasmic and nuclear extracts from normal human liver and two hepatocellular carcinoma cell lines, HepG2 and PLC/PRF/5, and compared the relative amounts of STX17 located in each fraction by Western blot analysis (Figure 8E). These data clearly demonstrate that the bulk of STX17 in normal hepatocytes is located in the cytoplasm in stark contrast to the situation in malignant cells, in which STX17 localization is reversed. To demonstrate the purity of the nuclear and cytoplasmic fractions, we then probed for PARP, a nuclear protein, in the same fractions (Figure 8F). These experiments confirm that there is little contamination between the nuclear and cytoplasmic fractions and give further credence to our results.
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Syntaxin 17 (STX17) is a divergent member of the syntaxin family of proteins, having been discovered in a yeast two-hybrid screen for proteins interacting with syntaxin 3 (Steegmaier et al. 1998
). In their characterization of STX17, Scheller and his colleagues reported that STX17 is ubiquitously expressed, but is expressed at particularly high levels in "steroidogenic" tissues such as adrenal gland, liver, ovary, placenta, and testis. Our experiments generally confirm these reports, but also point out discrepancies that prevent easy generalization about STX17's pattern of expression. In the adrenal gland, for example, there are four separate histologic zones with distinct functions: the zona glomerulosa, which synthesizes mineralocorticoids; the zona fasciculata, which synthesizes glucocorticoids; the zona reticulosa, which synthesizes sex hormones; and the adrenal medulla, which synthesizes catecholamines. Although the first three regions all produce steroid hormones and all four regions are secretory, both the zona glomerulosa and the adrenal medulla demonstrate significantly decreased STX17 expression compared with the other zones. In the testis, the male sex hormone–secreting Leydig cells are positive for STX17, but so also are Sertoli cells and germ cells within the seminiferous tubules. Finally, in the liver, hepatocytes do demonstrate STX17 expression, but levels of expression differ dramatically from cell to cell; the basis for these differences remains unclear, but is a subject of investigation. However, it is safe to say that there is no simple unifying theme to characterize those cells that express STX17 (e.g., secretory or steroidogenic). Prompted by these findings, we have conducted a more exhaustive survey of STX17 expression in various mouse and human tissues. Most notably, many surface epithelial cells exhibit strong STX17 expression, particularly squamous cells (e.g., skin, proximal esophagus, cervix/vagina) and urothelial cells lining the genitourinary tract. One also sees STX17 expressed elsewhere throughout the gastrointestinal tract. In the stomach, for example, both chief and parietal cells appear to express STX17, although a gradient of expression is noted with cells at the base of the gastric glands staining more strongly than those at the lumen. In the pancreas, STX17 is expressed at highest levels in islet and ductal epithelial cells with lesser expression noted in acinar cells. However, STX17 expression is generally decreased or absent in fibroblasts and other cell types found in the interstitium of most tissues.
In addition to the conclusion that STX17 can be strongly expressed in nonsteroidogenic cells, we have found that the subcellular localization of STX17 is heterogeneous and cell type–specific. STX17 can be localized to the cytoplasm (e.g., in hepatocytes, breast acinar epithelial cells, muscle cells, cells in the zona fasciculata and zona reticulosa of the adrenal gland, a subset of renal tubule cells) or to the nucleus. In fact, nuclear localization of STX17 can be demonstrated in a wide range of cells, including neurons; chondroblasts and osteoblasts; basilar chief and parietal cells in the stomach; basilar cells in intestinal crypts; Leydig, Sertoli, and germ cells in the testis; ductal and islet cells in the pancreas; and a subset of renal tubule cells and smooth and cardiac muscle cells. However, in addition to fibroblasts and other stromal cells, which typically lack STX17 expression, some epithelial and secretory cells also lack STX17 expression (e.g., alveolar epithelial cells and thyroid follicular cells), precluding easy generalization regarding the expression pattern of STX17.
We have also examined STX17 expression in hepatocellular and pancreatic adenocarcinoma cell lines. As expected based on the nuclear localization of STX17 in normal pancreatic ductal epithelial cells, STX17 is exclusively localized to the nucleus in four human pancreatic cancer cell lines. However, in contrast to the normal cytoplasmic localization of STX17 in hepatocytes, STX17 is exclusively localized to the nucleus in all three hepatocellular carcinoma cell lines tested. However, when we examined the pattern of STX17 expression in hepatocellular carcinomas isolated from patients at the time of surgery, we found that in the majority (five of six) of such cases, STX17 is localized primarily in the cytoplasm, the exception occurring in the only patient with a history of hepatitis B infection. These data serve as a cautionary note in extrapolating results obtained in human tumor cell lines passaged over many years to events in tumors in vivo and also raise interesting questions about the mechanism of intracellular localization of STX17 and the possible role of viruses in STX17 localization.
The mechanism by which STX17 is localized to various subcellular compartments within the cell is unknown. We have searched for a putative nuclear localization sequence on the basis of sequence homology with known consensus sequences, but have been unable to discern any such sequence. We are conducting experiments to address this issue, but the heterogeneous patterns of expression described here hint at a degree of complexity that will not be unraveled easily.
According to the SNARE hypothesis, syntaxins are thought to mediate membrane fusion events important in vesicular transport among various intracellular membrane compartments, and in keeping with this hypothesis, Steegmaier et al. have proposed that STX17 is involved in mediating such transport between the ER and Golgi (Steegmaier et al. 2000). However, the supporting evidence is circumstantial and primarily based on the localization of STX17 to the smooth ER in specific cell types and the physical association of STX17 with proteins, such as rbet1, rsec22b, and rsly1, that have been implicated in vesicular transport between the ER and Golgi (Zhang et al. 1997; Hay et al. 1998; Williams et al. 2004). Although not necessarily incompatible with these previous reports, our data suggest that STX17 may possess additional functions apart from a role in membrane trafficking. In particular, its nuclear localization in many cell types would be incompatible with its hypothesized function. For example, that STX17 is expressed in many cell types with high turnover (e.g., surface epithelial cells) and demonstrates altered nuclear localization in some tumor cells relative to their normal counterparts raises the possibility that STX17 may play a role in cell proliferation or transformation.
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Acknowledgments |
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We wish to thank Dr. Carolyn van Pelt and Wilfred Smith for their assistance in obtaining the archival mouse tissues and preparing the slides for IHC.
This work was supported by grants to L.H. from the National Institutes of Health (CA-71555), the Verto Institute, the Topfer Fund, the Eli Lilly & Co. Foundation Fund for Pancreatic Cancer Research, the M. D. Anderson Cancer Center, and by a P30 Cancer Center Support Grant (CA16672) to M. D. Anderson Cancer Center.
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Footnotes |
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Received for publication August 5, 2004; accepted May 11, 2005
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Literature Cited |
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Gana-Weisz M, Halaschek-Wiener J, Jansen B, Elad G, Haklai R, Kloog Y (2002) The Ras inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance. Clin Cancer Res 8:555–565
Hay JC, Klumperman J, Oorschot V, Steegmaier M, Kuo CS, Scheller RH (1998) Localization, dynamics, and protein interactions reveal distinct roles for ER and Golgi SNAREs. J Cell Biol 141:1489–1502
Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross S, et al. (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4:437–450[CrossRef][Medline]
Kita K, Saito S, Morioka CY, Watanabe A (1999) Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes. Int J Cancer 80:553–558[CrossRef][Medline]
Steegmaier M, Oorschot V, Klumperman J, Scheller RH (2000) Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics. Mol Biol Cell 11:2719–2731
Steegmaier M, Yang B, Yoo JS, Huang B, Shen M, Yu S, Luo Y, et al. (1998) Three novel proteins of the syntaxin/SNAP-25 family. J Biol Chem 273:34171–34179
Teng FY, Wang Y, Tang BL (2001) The syntaxins. Genome Biol 2:REVIEWS3012. Published online October 24
Williams AL, Ehm S, Jacobson NC, Xu D, Hay JC (2004) rsly1 binding to syntaxin 5 is required for endoplasmic reticulum-to-Golgi transport but does not promote SNARE motif accessibility. Mol Biol Cell 15:162–175
Zhang T, Wong SH, Tang BL, Xu Y, Peter F, Subramaniam VN, Hong W (1997) The mammalian protein (rbet1) homologous to yeast Bet1p is primarily associated with the pre-Golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol 139:1157–1168
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