点击显示 收起
【摘要】 The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) inhibits monocyte chemotactic protein-1- and stromal-derived factor-1 (SDF-1 )-mediated chemotaxis and diminishes chemokinesis in macrophage-like RAW264.7 and U937 cells in a manner that may involve attenuation of the intracellular calcium signal. STC1 is strongly induced in the kidney following obstructive injury. We hypothesized that STC1 may serve to attenuate the influx of inflammatory cells to the site of tissue injury. In this study, we examined the effect of STC1 on the migration of freshly isolated human macrophages, neutrophils, and T and B lymphocytes through quiescent or IL-1 -treated human umbilical vein endothelial cell (HUVEC) monolayers. STC1 inhibited transmigration of macrophages and T lymphocytes through quiescent or IL-1 -activated HUVECs but did not attenuate the transmigration of neutrophils and B lymphocytes. STC1 regulates gene expression in cultured endothelial cells and is detected on the apical surface of endothelial cells in vivo. The data suggest that STC1 plays a critical role in transendothelial migration of inflammatory cells and is involved in the regulation of numerous aspects of endothelial function.
【关键词】 array inflammation vascular biology calcium regulation
STANNIOCALCIN -1 (STC1) is a 25-kDa homodimeric glycoprotein hormone involved in calcium regulation in bony fish ( 17 ). Elevation of serum calcium in fish blood triggers the release of STC1 from the corpuscles of Stannius ( 45 ), organs associated with the kidneys ( 50 ). Upon circulation in the gill and intestine, STC1 inhibits calcium flux from the aquatic environment through these organs, thus maintaining stable concentrations of calcium in the blood ( 15, 31 ). In contrast to its restricted expression in bony fish, mammalian STC1 is expressed in many tissues and organs ( 7, 48 ) and does not normally circulate in the blood ( 49 ), and thus it is thought to function as an autocrine or paracrine substance. However, recent data suggest that mammalian STC1 may be carried by red blood cells and appears to be filtered through the glomeruli ( 25 ). Moreover, unlike its well-defined role in serum calcium regulation in fish, little is known about its function in mammals. Current data suggest, however, that STC1 may have a role in wound healing ( 24 ), cellular metabolism ( 34 ), atherogenesis ( 41 ), angiogenesis ( 26 ), steroidogenesis ( 38 ), muscle and bone development ( 16, 52 ), megakaryocyte differentiation ( 42 ), and cancer biology ( 8 ).
Functional analyses in mammals have suggested a role for STC1 in calcium regulation, as in vivo studies in the rat have suggested that STC1 may regulate serum calcium indirectly, by enhancing absorption of phosphate in the kidney ( 33 ), while in vitro studies in mammalian gut have demonstrated inhibition of calcium absorption and enhancement of phosphate uptake ( 33 ); in both instances, STC1 is predicted to decrease serum calcium. Therefore, through the evolutionary process from fish to mammals, STC1 appears to have maintained functional relevance to calcium homeostasis, while acquiring additional roles and functions in the various organs in which it is expressed.
Recent data from our laboratory provided additional evidence for the involvement of STC1 in calcium homeostasis in mammalian cells. Whole-cell patch-clamp studies in cardiomyocytes revealed inhibition of L-type calcium channels by STC1 ( 43 ). Moreover, STC1 diminishes the intracellular Ca 2+ (Ca ) signal in the macrophage-like cell line RAW264.7 in a dose-dependent manner and inhibits the response of monocyte/macrophage cell lines (Raw264.7 and U937) to chemokines ( 27 ). STC1 is strongly induced in the kidney following obstructive injury and is detected in the glomeruli, tubular epithelium, blood vessels, and macrophages ( 27 ), suggesting that STC1 may be an endogenous anti-inflammatory agent and leading us to hypothesize that STC1 may serve to attenuate the influx of inflammatory cells to the site of tissue injury. In the experiments reported here, we sought to determine the effect of STC1 on transendothelial migration of human neutrophils, macrophages, and T and B lymphocytes. Our current data suggest localization of STC1 at the apical surface of endothelial cells in vivo and reveal differential effects by STC1 on the migration of inflammatory cells across cultured endothelium. Some of these effects may be mediated through STC1-induced changes in endothelial function. In addition, STC1 induces wide-ranging changes in early (2 h) and late (24 h) gene expression in endothelial cells, and among the genes modified by STC1, we identified signaling molecules, transcription factors, mitochondrial proteins involved in redox and/or energy metabolism, ribosomal proteins and regulators of protein synthesis and degradation, cytoskeletal proteins and regulators of protein and/or organelle trafficking, cancer-related genes, cell cycle and apoptosis regulators, and, finally, cytokines and/or regulators of inflammation. These data suggest that STC1 is involved in the regulation of numerous aspects of endothelial function and plays a critical role in transendothelial migration of inflammatory cells at baseline, as well as during inflammation.
EXPERIMENTAL PROCEDURES
Materials. Human IL-1 was purchased from PeproTech (Rocky Hill, NJ). Recombinant hSTC1 protein was kindly provided by Dr. Henrik Olsen, Human Genome Sciences (Rockville, MD). It was expressed in a baculovirus 90% pure ( 12, 53 ). Endotoxin levels in STC1 preparation were determined using a Limulus Amebocyte Lysate Test Kit (Cambrex BioScience, Walkersville, MD) according to the manufacturer's instructions and showed no detectable endotoxin. Rabbit anti-trout STC1 antibodies were a generous gift from Dr. Gert Flik ( 51 ). Antibodies for human monocytes/macrophages (CD14), T lymphocytes (CD3), and B lymphocytes (CD20) were purchased from BD PharMingen (San Diego, CA). FITC-labeled monoclonal antibodies for hICAM-1 (CD54; clone BBIG-I1) and P-selectin (anti-CD62P; clone 9E1) were purchased from R&D Systems (Minneapolis, MN).
Endothelial cells. Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion as previously described ( 21 ) and used after a single passage ( P1 ). HUVEC monolayers were cultured in M199 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1% fungizone (Invitrogen), 1% HEPES buffer (Invitrogen), 1 µg/ml heparin (Sigma, St. Louis, MO), and endothelial cell growth supplement [ECGS; Fisher Scientific, Pittsburgh, PA]. After 4-6 days, HUVEC were passaged onto 35-mm tissue culture dishes (Corning Glass Works, Corning, NY), which were coated with glutaraldehyde-crosslinked gelatin, as described previously ( 5 ). HUVEC were used for the transmigration assays 2-4 days later.
Collection of macrophages, neutrophils, and B and T lymphocytes: Human neutrophils and mononuclear cells were obtained from peripheral blood samples using previously described methods ( 47 ). Briefly, citrated whole blood was sedimented by 1% dextran to remove red blood cells and centrifuged over a Ficoll gradient (Histopaque 1077, Sigma). Neutrophils were collected from the pellet and mononuclear cells (mixture of T lymphocytes, B lymphocytes, and macrophages) collected from the interphase, washed with 1 x PBS and used for assay. Remaining red blood cells in the neutrophil fraction were eliminated by quick hypotonic lysis in sterile water. The neutrophil fraction was 95% pure as evaluated 99% viable as evaluated by the trypan blue dye exclusion method. Neutrophils were kept at 4°C in Ca 2+ -free HEPES buffer (in mM: 110 NaCl, 10 KCl, 10 glucose, 1 MgCl 2, and 30 HEPES, pH 7.35) containing 0.1% human serum albumin (Armour Pharmaceuticals, Kankakee, IL). Lymphocytes were isolated from the mononuclear fraction using Lymphoprep solution (Axis-Shield, Oslo, Norway) according to the manufacturer's protocol. B lymphocytes and T lymphocytes were purified from the mixed lymphocyte preparation using a MagCellect* Human B lymphocytes Isolation Kit and CD3+ T lymphocytes Isolation Kit, respectively (R&D Systems) following the manufacturer's instructions.
Transendothelial migration. HUVEC monolayers on Transwell inserts were treated with IL-1 (10 U/ml) or vehicle for 4 h at 37°C in M199 media containing 4% FBS without ECGS or heparin. Inserts were placed over six-well plates coated with a thin layer of 1% agarose in 1 x PBS (the agarose layer facilitates removal of transmigrated cells for further analysis). Freshly isolated neutrophils, macrophages, and B or T lymphocytes (1 x 10 6 ) from healthy volunteers were added on the top chamber with a cells-to-HUVEC ratio of 2:1 and allowed to transmigrate through HUVEC for 1 (neutrophils), 2 (macrophages), or 24 h (T and B lymphocytes) at 37°C. The top chamber was removed, and the cell number in the bottom chamber was determined using a Z1 Beckman Coulter counter (Beckman Coulter, Fullerton, CA) or by flow cytometry using a FACS Vantage Flow cytometer (Becton Dickinson).
Measurement of Ca i 2 + by fluorescence spectrophotometry. Measurements of fluorescence intensity using a calcium fluoroprobe (Fluo 3, at 3 µM) and sequential image recordings were made on a Wallac/PerkinElmer (Gaithersburg, MD) Concord system incorporating a SpectraMaster multiwavelength controller and temperature-controlled stage (Melville, NY). To detail events over time, sequential image captures spanning 2-5 s were selected, and video recordings of these events were made for a number of minutes (average of 25,000 image acquisitions) using an Olympix AstroCam CCD4100 Fast Scan (12 bit; 768 x 576: 1,000 frames/s; 9-µm resolution).
SDS-PAGE. This method is based on Laemmli ( 30 ) with slight modifications. HUVECs were treated with STC1 (100 ng/ml) for 1 or 24 h, collected by centrifugation after scraping, and lysed in TLB [20 mM Tris (pH 7.4), 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM PMSF, and 1 µg/ml leupeptin]. Equal amounts of protein were resolved on 8% reducing SDS-PAGE. Proteins were transferred (4°C, 55 V, 3 h) onto Protran BA83 0.2-µm nitrocellulose membrane (Whatman, Sanford, ME) in Laemmli buffer (25 mM Tris, 52 mM glycine, pH 8.3). Blots were then blocked for 1 h at room temperature (RT) in PBST [50 mM NaPO 4 (pH 7.5), 100 mM NaCl, 0.05% Tween 20] containing 5% dry milk. This was followed by overnight incubation at 4°C with rabbit anti-KIF5B (a gift from Dr. Ronald Vale, UCSF) at a dilution of 1:1,000 (in PBST containing 5% dry milk), rabbit anti-speckle-type POZ protein (SPOP; Genway) at a dilution of 1:1,000, or rabbit anti-Daxx antibodies (Cell Signaling) at a dilution of 1:1,000. After a 25-min wash in PBST, blots were incubated for 1 h (RT) with peroxidase-conjugated secondary antibody, diluted (1:10,000) in PBST containing 5% milk. Blots were then washed for 25 min in PBST. and protein bands were visualized using the SuperSignal Enhancer detection system (Pierce, Rockford, IL) per the manufacturer's instructions. Band intensities were quantitated using Image Tool software (University of Texas Health Science Center, San Antonio, TX). Relative band intensities were normalized to -actin.
Determination of ICAM-1 and P-selectin expression on the surface of STC1-treated HUVEC. Cells were treated with STC1 (40, 100 or 500 ng/ml) for 15 min, 4 h, 16 h, or 24 h. HUVECs were then detached using 5 mM EDTA, washed, and treated with FITC-labeled human ICAM-1/CD54 monoclonal antibody (clone BBIG-I1; R&D Systems) or FITC-labeled P-selectin antibodies (anti-CD62P; Clone 9E1; R&D Systems). ICAM-1 and P-selectin expression was determined using flow cytometry (mean fluorescence).
Immunohistochemistry. Rat kidney tissue was fixed in 10% formaldehyde followed by dehydration in graded alcohols and embedding in paraffin blocks using standard techniques. Five-micrometer sections were cut, dried, and rehydrated for labeling with rabbit anti-trout STC1 serum (at a dilution of 1:500), using a peroxidase enzyme-based detection system (Vector Laboratories). Control for labeling was carried out in the presence of nonimmune rabbit serum and showed no staining. Photomicrographs were taken using a Labophot-2 Nikon microscope with a MagnaFire Olympus digital camera.
Gene array. Primary HUVEC (ATCC, Manassas, VA) were cultured in M199 medium containing the above-mentioned supplements. HUVEC monolayers were passaged after 4-6 days onto 10-cm tissue culture dishes (Corning Glass Works, Corning, NY), which were coated with glutaraldehyde-cross linked gelatin as described previously ( 5 ) and were used after a single or second passage ( P1-P2 ). After the cells reached confluence, quadruplicate HUVEC dishes were treated with recombinant human STC1 at a concentration of 100 ng/ml (concentrations within the physiological range of STC1 in various tissues) ( 13 ) for 2 or 24 h and total RNA was harvested for microarray studies using RNAzol (Tel-test, Friendswood, TX). Control samples were treated identically, except for omission of STC1.
DNase-treated RNA was amplified using a MessageAmp kit (Ambion, Austin, TX) according to the manufacturer's protocol. Amplification was carried out using 3 µg of total RNA, giving a yield of 50 µg RNA. Reverse transcription reactions were performed using EndoFree RT (Ambion) during which amino allyl dUTPs were incorporated. To increase the signal, reactions were run for 2 h at 42°C, contrary to the 48°C recommended for the RT enzyme. After 2 h, samples were denatured at 95°C for 5 min and immediately transferred to ice. Base hydrolysis of remaining RNA was performed by addition of 8.6 µl of 1 M NaOH and 8.6 µl of 0.5M EDTA, pH 8, and incubated at 65°C for 15 min. The solution was neutralized by the addition of 8.6 µl of 1 M HCl. Amino allyl-modified cDNA was purified using PCR purification columns (Qiagen, Valencia, CA). cDNA samples were brought up to 100 µl with Milli-Q water, to which 500 µl of buffer PB were added. The PCR purification protocol was followed exactly, with the exception of substituting 75% EtOH for buffer PE as a wash solution. cDNA was eluted off the column using 60 µl Milli-Q water, pH of 8.0, dried, and concentrated using speed-vac, followed by the addition of sodium bicarbonate (3 µl, 25 mg/ml). The cDNA was labeled with Alexa dyes via the free amine modification method (A-20002-546 Alexa Fluor and A-20006-647 Alexa Fluor, Molecular Probes, Eugene, OR). The tubes were then wrapped in foil and incubated at room temperature for 1 h. Each tube was brought up to 50 µl with Milli-Q water and combined. PCR purification columns (Qiagen) were used to carry out the same clean-up procedure as described for cDNA purification, except that one extra 75% EtOH wash was added and the final elution volume was 50 µl. An equal volume of hybridization buffer [8 x SSC (0.15M NaCl, 15 mM trisodium citrate, pH 7), 60% formamide, and 0.2% SDS] was added to the labeled cDNA. The sample was stored in foil until use.
Microarrays for our study were prepared within the Genomic Research Laboratory (GRL) at the University of Arizona using a combination of two human clone sets (10K and 12K), to yield a final printed array representing 22K clones. Clones were purchased from Research Genetics. Microarrays were prepared using custom-coated glass slides prepared using a 2% solution of 3-aminopropyltrimethoxysilane in 95% EtOH. In our experience, this coating procedure produces good attachment of DNA and an excellent signal-to-noise ratio (data not shown). DNA elements consist of purified PCR-amplified inserts utilizing primer pairs specific to the M13 sequences flanking each clone prepared in 100-µl reaction/clone. Amplification and purification of DNA inserts occur in a multiwell format, resulting in element DNA being suspended in 50% DMSO (300 ng/ml) in 384-well plates. Each clone is printed in a 45% humidified chamber using a Virtek ChipWriter microarray-printing robot equipped with 48-pin capability with individual element spots averaging 130 µm in diameter and separated by 160 µm center-to-center. Microarrays are stored in a desiccator at room temperature until ready for use. Two microarrays, randomly taken from the first and last 10 slides of the print run, are stained with the DNA dye SYBR Green II and scanned to qualify the print run for spot uniformity, array completeness, and minimum DNA content. These same microarrays are hybridized to labeled library DNA (from the library used to generate the element set) to ensure hybridization competency across the entire array. One microarray slide is removed from the desiccator for each hybridization and washed using a filtered 1% SDS solution heated to 60°C with moderate agitation for 2 min. The slide is then rinsed in Milli-Q water for 1 min and removed slowly to assure dryness and placed into the GeneTac hybridization station (Genome Solutions, Ann Arbor, MI). The labeled modified cDNA in the hybridization buffer is then loaded onto the slide and set to hybridize at 47°C for 16 h. After completion, a short wash is run in the hybridization station after which the slide is removed and dipped in 0.05 SSC to remove any residual nonhybridized cDNA. The slide is pulled out dry and analyzed using the arrayWoRxe CCD-based microarray scanner (Applied Precision, Issaquah, WA), which is capable of multichannel fluorescence scanning.
A multivariate experimental approach is used in the analysis of microarray data as previously published ( 6, 19, 35 ). This approach enables us to analyze a variety of variables in a microarray study (i.e., time course, treatment, condition, genotype, etc.) as well as identify all sources of experimental variance in the microarray data (i.e., array variation, dye performance, etc). This approach is based on an ANOVA statistical model (modified from G. Churchill: www.JAX.org ) with custom modifications to the best-fit algorithm for improved usability and increased applicability. This algorithm permits a robust characterization and classification of the data and provides outputs of residuals and other statistical parameters as references for users.
Individual samples within a group were labeled one through four. Each sample was hybridized within a treatment group to eliminate sample to sample variability. Each treatment group was hybridized four times, twice with each Alexa Fluor (see scheme below). Genes were deemed upregulated or downregulated if they demonstrated a minimum of 1.5-fold upward or downward shift (respectively) in gene expression that was detected consistently in three of four hybridization reactions above background, and more importantly significantly different from controls.
Statistical analysis. Data analysis for all studies (except for the array data) was carried out using Student's t -test and significance is reported at P < 0.05 using a commercially available statistical package (GraphPad PRISM 3.02).
RESULTS
STC1 inhibits macrophage transmigration through quiescent or IL-1 -treated HUVEC. Previous data from our laboratory suggested inhibition of the response of cultured human (U937) and murine (Raw264.7) macrophage-like cells to chemotactic stimulation by MCP1 or SDF-1 ( 27 ). In addition, STC1 was heavily induced in nearly all cellular elements in obstructed kidneys ( 27 ). These observations raised the question of whether upregulation of STC1 in obstructed kidneys served to retain inflammatory cells within the tissue or inhibit further flux of these cells to the site of injury. In the current experiments, we sought to determine the effects of STC1 on transmigration of human macrophages across endothelium. The number of migrating macrophages across quiescent endothelium was small (4%). Incubation of human macrophages with STC1 at a concentration (100 ng/ml; similar concentrations were reported to exist in vivo in many tissues including the kidneys) ( 13 ) that effectively inhibits chemotaxis in Raw264.7 and U937 cells ( 27 ), diminished macrophage transmigration across quiescent endothelium 75% ( Fig. 1 ). Macrophage transmigration across "activated" HUVEC (IL-1 -treated) was nearly 10-fold higher, and also, in this instance, STC1 diminished transmigration of macrophages to a similar extent ( 75%). To avoid nonspecific activation of macrophages by additional manipulations while separating T and B lymphocytes from the macrophages, these experiments were carried out using a mixture of mononuclear cells, containing macrophages, T and B lymphocytes, so a potential effect on macrophages, through interaction of STC1 with B or T lymphocytes, cannot be ruled out. However, based on previous data generated using U937 and Raw264.7 cells, where the effects of STC1 were direct, and notwithstanding the presence of T and B lymphocytes in the cell mixture, the observations made in our current experiments are likely the result of direct effects of STC1 on macrophages. Our current data suggest that STC1 may serve to diminish transendothelial migration of macrophages to injured tissue.
Fig. 1. Stanniocalcin-1 (STC1) inhibits macrophage transmigration through human umbilical vein endothelial cells (HUVEC). Freshly isolated mononuclear cells (a mixture of macrophages, B and T lymphocytes) were treated with recombinant endotoxin-free stanniocalcin-1 ( denotes denatured STC1) for 15 min and washed with 1 x PBS. Cells were allowed to transmigrate across quiescent or IL-1 -pretreated endothelium (10 U/ml for 4 h followed by wash in 1 x PBS), and the percentage of transmigrated macrophages was determined after 2 h by FACScan, using antibodies directed against CD14. Data shown represent the means of 3 separate experiments. * P < 0.05. ** P < 0.01.
STC1 inhibits T lymphocytes transmigration through activated HUVEC. We sought to determine the effects of STC1 on transmigration of purified neutrophils, T or B lymphocytes across the endothelium. Similar to the findings with macrophages, the number of transmigrating T lymphocytes across quiescent HUVEC was small (5%), while the number of transmigrating T lymphocytes across IL-1 -treated endothelium was 10-fold higher. As can be seen in Fig. 2, transendothelial migration of STC1-pretreated T lymphocyte was attenuated in a dose-dependent manner (75% inhibition at a concentration of 100 ng/ml); however, STC1 did not block the transmigration of neutrophils or B lymphocytes (data not shown). Thus STC1 exerts differential effects on the transmigration of inflammatory cells across endothelium, and we speculate that these differences relate to differential expression of STC1 receptors on these cells. As the migration of inflammatory cells across endothelium is influenced by the expression of adhesion molecules on endothelial cell surface ( 4, 40 ), we determined the expression of ICAM-1 and P-selectin expression in STC1-treated HUVEC using flow cytometry and found no significant changes (data not shown).
Fig. 2. STC1 inhibits T lymphocyte transmigration through stimulated HUVEC. Vehicle- or STC1-pretreated T lymphocytes (for 15 min followed by wash in 1 x PBS; denotes denatured STC1) were added atop quiescent or IL-1 -treated HUVEC (10 U/ml for 4 h followed by wash in 1 x PBS), and the number of transmigrated T lymphocytes was counted after 24 h. The percentage of transmigrated T lymphocytes is shown on the Y -axis. Data shown represent the means of 3 separate experiments. * P < 0.05 compared with IL-1 -treated cells.
STC1 increases Ca i 2 + in quiescent HUVEC and localizes to the apical surface of endothelial cells in vivo. Fluctuations in Ca concentrations have been linked to variations in endothelial cell function, such as the adhesion to, and transmigration of, inflammatory cells across the endothelium ( 4, 40 ). STC1 has been shown by our laboratory to diminish the Ca signal in cardiomyocytes ( 43 ) and murine Raw264.7 cells ( 27 ). Therefore, we sought to determine 1 ) whether STC1 is expressed on the surface of endothelial cells in vivo and 2 ) whether it affected the Ca signals in quiescent Fluo3-loaded HUVEC. As shown in Fig. 3, A and B, STC1 is detected on endothelial cells in the arteries (apical surface), venules, and glomerular capillaries of normal kidneys. Consistent with previously reported data ( 25 ), STC1 labeling was detected in red blood cells within arteries and venules ( Fig. 3, A and B ); however, STC1 was not present on the surface of all red blood cells. Of interest, and contrary to observations which we previously made in cardiomyocytes and Raw264.7 cells, where STC1 decreased the Ca signal in a dose-dependent manner ( 27, 43 ), low concentrations of STC1 (25-50 ng/ml) increased the Ca signal in endothelial cells ( Fig. 3 D ), while higher concentrations of the protein ( 200 ng/ml) had no significant effect (data not shown). The rise in the Ca signal occurred within 2 min following the addition of STC1 10 min. Our data demonstrate expression of STC1 in endothelial cells under normal conditions in vivo and suggest a role for this molecule in regulating endothelial function.
Fig. 3. A - C : STC1 labeling is detected on endothelial cells of normal kidney arteries, venules, and glomerular capillaries. Art, artery; G, glomerulus; arrowheads point to venules in the cortex ( A ) and glomerular capillaries ( B ). Magnification x 500. C : nonimmune serum shows no labeling. D : STC1 increases intracellular Ca 2+ (Ca signal in HUVEC. HUVEC were loaded with fluo 3 and then treated with STC1. STC1 (50 ng/ml) increased Ca signal. Ca appeared increased within 2-3 min after application of STC1 to the cells and remained at a high plateau for several minutes. Shown are simultaneous Ca tracings from 4 separate cells in the camera view.
Regulation of gene expression in endothelial cells by STC1. The detection of STC1 on the apical surface of endothelial cells in vivo and the effects of STC1 on Ca in HUVEC suggested that STC1 may affect gene expression and function in endothelial cells. In the following experiment, we sought to determine the effect of STC1 on gene expression in quiescent HUVEC, using a human gene array chip representing 22,000 genes (Genomic Research Laboratory, University of Arizona). Treatment of HUVEC with STC1 for 2 and 24 h induced significant changes in the expression of a large number of genes (some were upregulated, while the others were downregulated; see Table 1 ). These included genes coding for signaling molecules, transcription factors, cancer-related genes, regulators of cell cycle and apoptosis, inflammation-related molecules, ribosomal proteins and regulators of protein synthesis and/or degradation, cytoskeletal proteins and regulators of protein and/or organelle trafficking, and finally proteins involved in redox or mitochondrial energy metabolism. Two of the genes identified by the array are related to current work on STC1, and thus we determined the expression of the corresponding proteins using Western blotting; these include the kinesin family member 5B (KIF5B; accession no. AA046690 ) and SPOP (accession no. R35231 ); both were upregulated at 2 and 24 h. Data in the literature suggest that KIF5B is important for the organization of mitochondrial DNA and for mitochondrial trafficking ( 22, 46 ), and recent data from our laboratory implicated STC1 in the regulation of uncoupling protein 2 (UCP2) and superoxide production in macrophage mitochondria (unpublished observations). Treatment of HUVEC with STC1 for 24 h increased KIF5B protein levels twofold ( Fig. 4 A ), consistent with a role for STC1 in mitochondrial organogenesis and function. SPOP, on the other hand, serves as an adaptor for Daxx and facilitates the ubiquitination and degradation of Daxx by Cul3-based ubiquitin ligase ( 28, 29 ). Daxx is an adaptor molecule that links death receptors to caspase-8 activation in many cell types and has been implicated in the regulation of NF- B-mediated transcription ( 9, 36 ) and hence its relevance to inflammation. As can be seen in Fig. 4 B, treatment of HUVEC with STC1 upregulates SPOP and downregulates Daxx (at 24-h time point). Thus, by inducing changes in SPOP, STC1 may have a considerable impact on Daxx-related cellular processes including cell survival and NF- B signaling. These will be addressed in future studies.
Table 1. Gene array data from STC1-treated HUVEC
Fig. 4. A : protein lysates from vehicle- or STC1-treated HUVEC were resolved on 8% SDS-PAGE, and blots were reacted with anti-KIF5B or anti- -actin antibodies. Representative blot is shown, while bar graph depicts KIF5B band intensities, normalized to -actin, and represents the means of 3 separate experiments. * P < 0.05 compared with vehicle-treated cells. B : protein lysates from vehicle- or STC1-treated HUVEC were resolved on 8% SDS-PAGE, and blots were reacted with anti-SPOP, anti-Daxx, or anti- -actin antibodies. Representative blot is shown.
DISCUSSION
The role of STC1 in the immune system and inflammation is currently unknown. Previous data from our lab suggested that STC1 is a novel, naturally occurring blocker of L-type calcium channels in cardiomyocytes ( 43 ) and an inhibitor of monocyte function ( 27 ); it diminishes the Ca signal in cultured monocytes and inhibits the response of two monocyte cell lines (murine Raw264.7 and human U937 cells) to MCP1 and SDF-1 ( 27 ). In addition, STC1 diminishes the random movement of monocytes independently of chemokine gradient-directed motion. These data suggest that through evolution from fish to mammals, STC1 has retained its effects on calcium metabolism, functioning in fish as a true calcium-regulatory hormone, being produced in one organ (the gland of Stannius) ( 45 ) and functioning in another (the gut and intestine), while in mammals it is ubiquitously expressed and acts in an autocrine/paracrine manner ( 23, 32, 52 ). In addition to its involvement in calcium homeostasis, STC1 has acquired new functions in mammals, and this paper discusses one of these functions.
Inhibition of chemokinesis by STC1 suggested a general mechanism of action, independently of the chemokine/receptor pathway ( 27 ). MCP1 and SDF-1 operate through distinct receptors (CCR2 and CXCR4, respectively) ( 54 ), also suggesting that STC1 acts on a common pathway distal to receptor stimulation by the chemokine, possibly through attenuation of the Ca signal. The effects of STC1 on cultured monocytes are physiologically/pathophysiologically relevant, as STC1 is strongly induced in the kidney following unilateral ureteric obstruction ( 27 ), which is a model of inflammatory injury ( 14, 39 ). In addition, labeling studies have revealed localizations of STC1 to macrophages/monocytes in obstructed kidneys ( 27 ). Our hypothesis was that upregulation of STC1 in the obstructed kidney serves to attenuate the influx of inflammatory cells to the site of the injury, and so to test this, we examined the migration of freshly isolated human inflammatory cells (macrophages, neutrophils, T and B lymphocytes) across quiescent or activated (IL-1 -treated) cultured primary HUVEC. STC1 diminishes the migration of macrophages and T lymphocytes across quiescent or activated endothelial cells but does not inhibit transmigration of neutrophils or B lymphocytes. These data are consistent with the effects of STC1 on monocyte cell lines that we previously reported ( 27 ) and suggest an important role for STC1 in modulating inflammatory cell function both under basal conditions and during inflammation. We speculate that the observed differential effects of STC1 on macrophages and T lymphocytes, as opposed to neutrophils and B lymphocytes, may be related to differential expression of STC1 receptors. In addition, our data suggest expression of STC1 in blood vessels (arterioles, venules, and glomerular capillaries) of normal kidney and apparent localization to the apical surface of endothelial cells (at least in arterioles). Our findings support a role for STC1 in transendothelial migration of macrophages and T lymphocytes in vivo, suggesting that STC1 may regulate inflammation at multiple levels 1 ) through direct action on inflammatory cells and 2 ) through modulation of endothelial cell function and its response to cytokines. Indeed, gene array data demonstrate that STC1 exerts both short-term (2 h) and long-term (24 h) effects on gene expression in cultured HUVEC, regulating a number of genes in endothelial cells that "fit" in distinct "gene profiles," most notably being genes that are relevant to cell growth and differentiation (growth factors and regulators of cell cycle and apoptosis); genes whose expression is altered in various tumors, particularly colon cancer, lymphomas, and malignancies of the central nervous system and kidneys; genes important for ribosomal structure/function; genes relevant to energy metabolism and mitochondrial function, a large number of transcription factors, some of which are relevant to vascular biology and hematopoiesis, serine/threonine kinases, and their respective phosphatases; genes involved in cytoskeletal organization and organelle trafficking; and genes involved in inflammation. Thus STC1 may play a role in the normal physiology of endothelial cells as well as endothelial processes underlying the pathophysiology of a number of disease processes that include, but may not be limited to, inflammation, vascular biology, energy metabolism, and cancer.
Activated endothelial cells express cell surface adhesion molecules, such as ICAM-1, and selectins, which contribute to the recruitment of mononuclear cells and facilitate their migration into the subendothelial space ( 3, 44 ). As shown above, the expression of ICAM-1 and P-selectin in endothelial cells was not altered by STC1, so it needs to be determined whether STC1 alters endothelial barrier function during inflammation, which is dependent on the expression of junction proteins ( 1, 2, 10, 18, 37 ), or whether STC1 alters cytokine-induced signaling pathways, such as reactive oxygen species, NF- B, and stress kinases (p38 kinase and JNK in endothelial cells), in the setting of cytokine exposure ( 11, 20 ). These questions will be addressed in future studies.
In summary, our data suggest that STC1 is an immune modulator and possesses complex effects on inflammatory cells, inhibiting the influx of macrophages and T lymphocytes but not B lymphocytes or neutrophils and altering gene expression in endothelial cells in a manner that may fundamentally affect the normal physiology of these cells as well as affect processes underlying the pathophysiology of numerous disease processes that include inflammation to vascular biology, energy metabolism, and cancer.
GRANTS
This work was supported by O'Brien Kidney Center Grant DK-064233-01.
【参考文献】
Anderson JM, Balda MS, Fanning AS. The structure and regulation of tight junctions. Curr Opin Cell Biol 5: 772-778, 1993.
Anderson JM, Van Itallie CM. Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol Gastrointest Liver Physiol 269: G467-G475, 1995.
Aplin AE, Howe A, Alahari SK, Juliano RL. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev 50: 197-263, 1998.
Blaheta RA, Hailer NP, Brude N, Wittig B, Oppermann E, Leckel K, Harder S, Scholz M, Weber S, Encke A, Markus BH. Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium. Immunology 94: 213-220, 1998.
Burns AR, Bowden RA, Abe Y, Walker DC, Simon SI, Entman ML, Smith CW. P-selectin mediates neutrophil adhesion to endothelial cell borders. J Leukoc Biol 65: 299-306, 1999.
Cai Q, Keck M, McReynolds MR, Klein JD, Greer K, Sharma K, Hoying JB, Sands JM, Brooks HL. Effects of water restriction on gene expression in mouse renal medulla: identification of 3 HSD4 as a collecting duct protein. Am J Physiol Renal Physiol 291: F218-F224, 2006.
Chang AC, Janosi J, Hulsbeek M, de Jong D, Jeffrey KJ, Noble JR, Reddel RR. A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol Cell Endocrinol 112: 241-247, 1995.
Chang AC, Jellinek DA, Reddel RR. Mammalian stanniocalcins and cancer. Endocr Relat Cancer 10: 359-373, 2003.
Chao W, Shen Y, Li L, Zhao H, Meiler SE, Cook SA, Rosenzweig A. Fas-associated death-domain protein inhibits TNF- -mediated NF- B activation in cardiomyocytes. Am J Physiol Heart Circ Physiol 289: H2073-H2080, 2005.
Chavakis T, Preissner KT, Santoso S. Leukocyte trans-endothelial migration: JAMs add new pieces to the puzzle. Thromb Haemost 89: 13-17, 2003.
Chen XL, Zhang Q, Zhao R, Medford RM. Superoxide, H 2 O 2, and iron are required for TNF- -induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase. Am J Physiol Heart Circ Physiol 286: H1001-H1007, 2004.
De Niu P, Olsen HS, Gentz R, Wagner GF. Immunolocalization of stanniocalcin in human kidney. Mol Cell Endocrinol 137: 155-159, 1998.
De Niu P, Radman DP, Jaworski EM, Deol H, Gentz R, Su J, Olsen HS, Wagner GF. Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat. Mol Cell Endocrinol 162: 131-144, 2000.
Diamond JR, Kees-Folts D, Ding G, Frye JE, Restrepo NC. Macrophages, monocyte chemoattractant peptide-1, and TGF- 1 in experimental hydronephrosis. Am J Physiol Renal Fluid Electrolyte Physiol 266: F926-F933, 1994.
Fenwick JC, Brasseur JG. Effects of stanniectomy and experimental hypercalcemia on plasma calcium levels and calcium influx in American eels, Anguilla rostrata LeSueur. Gen Comp Endocrinol 82: 459-465, 1991.
Filvaroff EH, Guillet S, Zlot C, Bao M, Ingle G, Steinmetz H, Hoeffel J, Bunting S, Ross J, Carano RA, Powell-Braxton L, Wagner GF, Eckert R, Gerritsen ME, and French DM. Stanniocalcin 1 alters muscle and bone structure and function in transgenic mice. Endocrinology 143: 3681-3690, 2002.
Fontaine M. Corpuscles de Stannius et regulation ionique (Ca, K, Na) du Milieu interieur de l'Anguille ( Anguilla anguilla L.). C R Acad Sc Paris Serie D 259: 875-878, 1964.
Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 1617-1626, 1994.
Greer KA, McReynolds MR, Brooks HL, Hoying JB. CARMA: a platform for analyzing microarray datasets that incorporate replicate measures. BMC Bioinformatics 7: 149, 2006.
Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 20: 2175-2183, 2000.
Huang AJ, Furie MB, Nicholson SC, Fischbarg J, Liebovitch LS, Silverstein SC. Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules. J Cell Physiol 135: 355-366, 1988.
Iborra FJ, Kimura H, Cook PR. The functional organization of mitochondrial genomes in human cells. BMC Biol 2: 9, 2004.
Ishibashi K, Imai M. Prospect of a stanniocalcin endocrine/paracrine system in mammals. Am J Physiol Renal Physiol 282: F367-F375, 2002.
Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JC, Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D, Shalon D, Botstein D, Brown PO. The transcriptional program in the response of human fibroblasts to serum. Science 283: 83-87, 1999.
James K, Seitelbach M, McCudden CR, Wagner GF. Evidence for stanniocalcin binding activity in mammalian blood and glomerular filtrate. Kidney Int 67: 477-482, 2005.
Kahn J, Mehraban F, Ingle G, Xin X, Bryant JE, Vehar G, Schoenfeld J, Grimaldi CJ, Peale F, Draksharapu A, Lewin DA, Gerritsen ME. Gene expression profiling in an in vitro model of angiogenesis. Am J Pathol 156: 1887-1900, 2000.
Kanellis J, Bick R, Garcia G, Truong L, Tsao CC, Etemadmoghadam D, Poindexter B, Feng L, Johnson RJ, Sheikh-Hamad D. Stanniocalcin-1, an inhibitor of macrophage chemotaxis and chemokinesis. Am J Physiol Renal Physiol 286: F356-F362, 2004.
Kwon JE, La M, Oh KH, Oh YM, Kim GR, Seol JH, Baek SH, Chiba T, Tanaka K, Bang OS, Joe CO, Chung CH. BTB domain-containing speckle-type POZ protein (SPOP) serves as an adaptor of Daxx for ubiquitination by Cul3-based ubiquitin ligase. J Biol Chem 281: 12664-12672, 2006.
La M, Kim K, Park J, Won J, Lee JH, Fu YM, Meadows GG, Joe CO. Daxx-mediated transcriptional repression of MMP1 gene is reversed by SPOP. Biochem Biophys Res Commun 320: 760-765, 2004.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.
Lafeber FP, Flik G, Wendelaar Bonga SE, Perry SF. Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. Am J Physiol Regul Integr Comp Physiol 254: R891-R896, 1988.
Luo CW, Kawamura K, Klein C, Hsueh AJ. Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin 1 (STC1): mediation through specific STC1 receptors. Mol Endocrinol 18: 2085-2096, 2004.
Madsen KL, Tavernini MM, Yachimec C, Mendrick DL, Alfonso PJ, Buergin M, Olsen HS, Antonaccio MJ, Thomson AB, Fedorak RN. Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine. Am J Physiol Gastrointest Liver Physiol 274: G96-G102, 1998.
McCudden CR, James KA, Hasilo C, Wagner GF. Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism. J Biol Chem 277: 45249-45258, 2002.
McReynolds MR, Taylor-Garcia KM, Greer KA, Hoying JB, Brooks HL. Renal medullary gene expression in aquaporin-1 null mice. Am J Physiol Renal Physiol 288: F315-F321, 2005.
Michaelson JS, Leder P. RNAi reveals anti-apoptotic and transcriptionally repressive activities of DAXX. J Cell Sci 116: 345-352, 2003.
Ostermann G, Weber KS, Zernecke A, Schroder A, Weber C. JAM-1 is a ligand of the beta 2 integrin LFA-1 involved in transendothelial migration of leukocytes. Nat Immunol 3: 151-158, 2002.
Paciga M, McCudden CR, Londos C, DiMattia GE, Wagner GF. Targeting of big stanniocalcin and its receptor to lipid storage droplets of ovarian steroidogenic cells. J Biol Chem 278: 49549-49554, 2003.
Ricardo SD, Diamond JR. The role of macrophages and reactive oxygen species in experimental hydronephrosis. Semin Nephrol 18: 612-621, 1998.
Saito H, Minamiya Y, Kitamura M, Saito S, Enomoto K, Terada K, Ogawa J. Endothelial myosin light chain kinase regulates neutrophil migration across human umbilical vein endothelial cell monolayer. J Immunol 161: 1533-1540, 1998.
Sato N, Kokame K, Shimokado K, Kato H, Miyata T. Changes of gene expression by lysophosphatidylcholine in vascular endothelial cells: 12 up-regulated distinct genes including 5 cell growth-related, 3 thrombosis-related, and 4 others. J Biochem (Tokyo) 123: 1119-1126, 1998.
Serlachius M, Alitalo R, Olsen HS, Andersson LC. Expression of stanniocalcin-1 in megakaryocytes and platelets. Br J Haematol 119: 359-363, 2002.
Sheikh-Hamad D, Bick R, Wu GY, Christensen BM, Razeghi P, Poindexter B, Taegtmeyer H, Wamsley A, Padda R, Entman M, Nielsen S, Youker K. Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure. Am J Physiol Heart Circ Physiol 285: H442-H448, 2003.
Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 57: 827-872, 1995.
Stannius H. Uber nebenniere bei knochenfischen. Arch Anat Physiol 6: 97-101, 1939.
Tanaka Y, Kanai Y, Okada Y, Nonaka S, Takeda S, Harada A, Hirokawa N. Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria. Cell 93: 1147-1158, 1998.
Tkatch LS, Rubin KA, Ziegler SF, Tweardy DJ. Modulation of human G-CSF receptor mRNA and protein in normal and leukemic myeloid cells by G-CSF and retinoic acid. J Leukoc Biol 57: 964-971, 1995.
Varghese R, Wong CK, Deol H, Wagner GF, DiMattia GE. Comparative analysis of mammalian stanniocalcin genes. Endocrinology 139: 4714-4725, 1998.
Wagner GF, De Niu P, Jaworski E, Radman D, Chiarot C. Development of a dose-response bioassay for stanniocalcin in fish. Mol Cell Endocrinol 128: 19-28, 1997.
Wendelaar Bonga SE, Pang PK. Control of calcium regulating hormones in the vertebrates: parathyroid hormone, calcitonin, prolactin, and stanniocalcin. Int Rev Cytol 128: 139-213, 1991.
Wendelaar Bonga SE, Smits PW, Flik G, Kaneko T, Pang PK. Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout, flounder and goldfish). Cell Tissue Res 255: 651-656, 1989.
Yoshiko Y, Aubin JE, Maeda N. Stanniocalcin 1 (STC1) protein and mRNA are developmentally regulated during embryonic mouse osteogenesis: the potential of stc1 as an autocrine/paracrine factor for osteoblast development and bone formation. J Histochem Cytochem 50: 483-492, 2002.
Zhang J, Alfonso P, Thotakura NR, Su J, Buergin M, Parmelee D, Collins AW, Oelkuct M, Gaffney S, Gentz S, Radman DP, Wagner GF, Gentz R. Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells. Protein Expr Purif 12: 390-398, 1998.
Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127, 2000.
作者单位：3 Renal Section, Department of Medicine, and 1 Leukocyte Biology, Children‘s Nutritional Research Center, Baylor College of Medicine, 4 Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, and 5 Department of Renal Pathology, Methodist Hospital, Houston,