点击显示 收起
【摘要】 In macrophages, changes in intracellular calcium have been associated with activation of cellular processes that regulate cell adhesion and motility and are important for the response of macrophages to antigenic stimuli. The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in multiple organs including the thymus and spleen, and hence, we hypothesized that it may have a role in modulating the immune/inflammatory response. Using murine macrophage-like (RAW264.7) and human monoblast-like (U937) cells to study chemotaxis in vitro, we found that STC1 attenuated chemokinesis and diminished the chemotactic response to monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1. Consistent with these findings, STC1 blunted the rise in intracellular calcium following MCP-1 stimulation in RAW264.7 cells. In vivo studies suggested differential expression of STC1 in obstructed kidney and localization to macrophages. MCP-1 and STC1 transcripts were both upregulated following ureteric obstruction, suggesting a functional association between the two genes. Our data suggest a role for mammalian STC1 in modulating the immune/inflammatory response.
【关键词】 monocyte chemotactic protein stromal cellderived factor ureteric obstruction calcium homeostasis kidney injury
CALCIUM FLUX ACROSS the plasma membrane and its release from intracellular stores play a major role in chemotaxis in macrophages and other inflammatory cells. In macrophages, intracellular calcium ([Ca 2+ ] i ) changes have been associated with activation of cellular kinases and phosphatases, degranulation, phagosome-lysosome fusion, cytoskeletal reorganization, transcriptional control, modulation of surface receptors, and regulation of cell adhesion and motility ( 16 ), processes that are important for the cellular response to antigenic stimuli. In lymphocytes, calcium plays a key role in chemotaxis, where [Ca 2+ ] i signals contain a wealth of information that may instruct the cell to choose between divergent fates in response to antigenic stimuli, such as proliferation and/or apoptosis ( 14 ). In the short term, [Ca 2+ ] i signals help stabilize the contacts between T-lymphocytes and antigen-presenting cells through changes in motility and cytoskeletal reorganization, while in the long term (minutes to hours), [Ca 2+ ] i signals determine gene activation ( 14 ).
Stanniocalcin-1 (STC1) is a calcium-regulating hormone in bony fish ( 8 ), where elevation of serum calcium triggers the release of STC1 from the corpuscles of Stannius ( 21 ), organs associated with the kidneys ( 27 ). Upon circulation in the gill and intestine, STC1 inhibits calcium flux from the aquatic environment through these organs, thus maintaining stable calcium concentrations in the blood ( 12, 22 ). In mammals, STC1 is expressed in multiple organs, including the brain, thyroid, spleen, thymus, parathyroid, lung, heart, skeletal muscle, kidney, pancreas, small intestine, colon, placenta, ovary, testes, and prostate ( 1, 2, 20 ). The wide expression of STC1 suggested that it might function in an autocrine and/or paracrine manner, while its localization to the thymus and spleen suggested a role in the immune/inflammatory response. Furthermore, through the evolutionary process from fish to mammals, STC1 appears to have maintained functional relevance to calcium regulation, as mammalian STC1 is involved in calcium homeostasis in the normal physiology of the gut ( 15 ) and in the adaptive response of brain cells to ischemic injury ( 30 ).
We hypothesized that, in macrophages, as in gut and brain cells, STC1 may affect calcium flux across cellular membranes, thereby leading to modulation of the inflammatory/immune response.
METHODS
Materials. All chemicals were purchased from Sigma Chemical (St. Louis, MO), except where stated. Recombinant rat monocyte chemotactic protein-1 (MCP-1) and recombinant human stromal cell-derived factor-1 (SDF-1 ) were purchased from BD Pharmingen (San Diego, CA). STC1-specific rabbit polyclonal antibodies (that do not cross-react with STC2) and recombinant hSTC1 protein [expressed in a baculovirus expression system and is greater than 90% pure ( 6, 29 )] were kindly provided by Dr. H. Olsen, Human Genome Sciences (Rockville, MD).
Unilateral ureteric obstruction in the mouse and rat. Under methoxyflurane inhalation anesthesia, unilateral ureteric obstruction (UUO) was created in 12-wk-old C57BL/6 mice by complete ligation of the left ureter at the ureteropelvic junction using double-silk sutures. A similar procedure was used to create UUO in the rat (male Sprague-Dawley rats; 200-g initial weight; Harlan Bioproducts for Science). Animals were placed on regular diet, allowed free access to tap water, and killed at varying time points (on days 4, 7, 15, 21, and 31; 3 animals per time point). These time points were chosen as they were shown in previous studies to reflect early and midpoint changes in the evolution of renal injury following ureteric obstruction ( 23, 24 ). Obstructed, nonligated-contralateral or sham-operated kidneys were processed for immunohistochemistry or snap-frozen in liquid nitrogen and stored at -70°C until used. The experimental protocols were performed in accordance with Baylor College of Medicine animal care committee.
Immunohistochemistry. Kidney tissue (mouse and rat) was fixed in 10% formaldehyde followed by dehydration in graded alcohols and embedded in paraffin blocks using standard techniques. Five-micrometer sections were cut, dried, and rehydrated for labeling with affinity-purified anti-hSTC1 antibodies, using peroxidase enzyme-based detection system (Vector Laboratories). Control for labeling was carried out in the presence of normal IgG and showed no staining. Colocalization of STC1 and macrophage markers (using anti-hSTC1 and mouse anti-rat monocyte/macrophage/dendritic cell clone ED1; Research Diagnostics, Flanders, NJ) was demonstrated using a dual-labeling kit (Zymed, San Francisco, CA). Photomicrographs were taken using Labophot-2 Nikon microscope with a MagnaFire Olympus digital camera.
Measurement of [Ca 2+ ] i by fluorescence spectrophotometry. Measurements of fluorescence intensity of calcium fluoroprobe (Fluo3 at 3 µM) and sequential image recordings were made on a Wallac/Perkin Elmer (Gaithersburg, MD) Concord system incorporating a SpectraMaster multi-wavelength 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).
Chemotaxis and chemokinesis assays. Murine macrophage-like (RAW264.7) and human monoblast-type (U937) cells were used to assess the effects of STC1 on both random cell movement (chemokinesis) and chemokine-mediated chemotaxis. RAW264.7 cells were maintained in DMEM. U937 cells were maintained in RPMI. Both media were supplemented with 10% FBS. RAW264.7 cells are adherent cells, whereas U937 cells grow in suspension. Once confluent, RAW264.7 cells were passaged and resuspended at a concentration of 2 x 10 6 cells/ml in DMEM with 10% FBS. U937 cells in suspension were pelleted and resuspended in RPMI with 10% FBS at the same concentration (2 x 10 6 cells/ml). Both cell types were then incubated with STC1 (0, 10, 50, 100, 500 ng/ml) just before the assays. Denatured STC1 (boiled for 5 min) at the same concentration was used as a negative control.
Chemotaxis and chemokinesis were measured using either transfilter (cells trapped in pores-RAW264.7 cells) or transwell assays (cells reaching lower chamber-U937 cells) in 48-well chemotaxis chambers (Neuroprobe, Cabin John, MD), as previously described ( 3, 28 ). Media with or without recombinant rat MCP-1 (200 ng/ml; BD Pharmingen) or SDF-1 (150 ng/ml; BD Pharmingen) was placed in lower wells of chambers (30 µl) and separated from the cell suspension (50 µl) in the upper wells by polyvinylpyrrolidone-free polycarbonate filters (8- or 5-µm pore size). Chemotaxis chambers were incubated at 37°C in 5% CO 2 for 90 min. In the transfilter assays (RAW264.7 cells), the filter membrane upper surface was subsequently washed three times with PBS and scraped to remove cells, which had settled on the membrane upper surface. Cells trapped in the filter pores or adherent to the undersurface were fixed in methanol, stained with Kwik-Diff (Thermo Shandon, Pittsburgh, PA), and counted. The total number of cells trapped in the pores was assessed for each well. In the transwell assays (U937 cells), the total number of cells reaching the lower well was counted using a hemacytometer. Counts were expressed as a percentage of the total number of cells added to the upper chamber. For both transfilter and transwell assays, each combination of upper and lower well configurations was performed in quadruplicate.
Ribonuclease protection assay. Total RNA was extracted using RNazol B (Tel-Test, Friendswood, TX) according to previously published protocols ( 4 ). Ten micrograms of total RNA were subjected to ribonuclease protection assay using RiboQuant kit and mCK-5 cDNA template set (Pharmingen). The cDNA template set used contained multiple probes, which allowed simultaneous detection of mRNAs corresponding to various inflammatory mediators (Ltn, RANTES, Eotaxin, MIP-1, MIP-1, MIP-2, IP-10, MCP-1, TCA-3). cDNA probes for ribosomal protein L32 and glyceraldehyde-3-phosphate dehydrogenase were included as internal controls. In a separate ribonuclease protection assay, we examined the expression of STC1 and MCP-1 mRNAs in obstructed mouse kidneys, using murinespecific probes for STC1 (bp 1-200) and MCP-1 (bp 20-160). Both probes were sublconed into pGEM4Z with the 3' end of the cDNAs toward T7 promoter. Radiolabeled antisense RNA probes were synthesized using an in vitro transcription system (Promega, Madison, WI) and [ - 32 P]UTP (3,000 Ci/mmol; ICN). Ribonuclease protection assay was performed on 4 µg of total RNA using ribonuclease protection assay kit I (Torrey Pines Biolabs) according to the manufacturer's instructions. Antisense RNA probes were hybridized with the RNA samples at 90°C for 25 min. Unhybridized single-strand RNA was digested using Ribonuclease A/T1 (Sigma) for 30 min. Double-strand RNA was precipitated using stop solution at -80°C for 15-30 min and centrifuged at 15,000 g for 30 min. The samples were resolved on 6% sequencing gel. The gels were dried and exposed to X-ray films.
Statistical analysis. Results were expressed as means ± SD. Statistical significance was assessed using t -test or ANOVA and the Bonferroni multiple comparison test. P values <0.05 were considered statistically significant.
RESULTS
STC1 attenuates MCP-1-mediated [Ca 2+ ] i signal in the macrophage-like RAW264.7 cells. In macrophages, occupancy of the chemokine receptor by its ligand leads to calcium mobilization from intracellular compartments through a number of potential pathways [mediated by inositol 1,4,5P 3 (IP 3 ), ryanodine, or sphingosine-1-phosphate (S1P); ( 9, 13, 18, 31 )]. Using murine macrophage-like RAW264.7 cells, we assessed the effect of STC1 on MCP-1-mediated [Ca 2+ ] i alterations. Treatment of the cells with STC1 attenuated [Ca 2+ ] i in a dose-dependent manner, with half-maximal response observed at a concentration of 100 ng/ml ( Fig. 1 ), and hence, we used this concentration for most of the incubations. However, peak effect of STC1 on [Ca 2+ ] i was observed at 500 ng/ml. As expected, addition of MCP-1 to RAW264.7 cells increased cytoplasmic calcium concentrations (measured as Fluo3 fluorescence) within minutes ( Fig. 1 ). Pretreatment of the cells with STC1, on the other hand, decreased baseline [Ca 2+ ] i and blunted the rise in cytoplasmic calcium in response to MCP-1 treatment. In control experiments, addition of heat-denatured STC1 or STC1-neutralizing antibodies plus STC1 had no effect on MCP-1-mediated rise in [Ca 2+ ] i (data not shown). These findings suggest that STC1 affects calcium homeostasis in activated macrophages, and as a consequence, potentially modifies the immune/inflammatory response.
Fig. 1. Stanniocalcin-1 (STC1) attenuates intracellular calcium concentration ([Ca 2+ ] i ) and inhibits [Ca 2+ ] i rise in response to monocyte chemotactic protein-1 (MCP-1) treatment in RAW264.7 cells. RAW264.7 cells were grown on cover slips and maintained in DMEM supplemented with 10% FBS. Subconfluent cells (5 x 10 6 /ml) were incubated with Fluo3 ([Ca 2+ ] i fluorescence probe; 3 µM) at 37°C for 15 min and washed before the addition of experimental reagents. Extracellular Ca 2+ concentration was maintained at 1 mM. A : [Ca 2+ ] i in response to increasing concentrations of recombinant STC1 (20, 50, 100, and 500 ng/ml); y -axis depicts [Ca 2+ ] i values, as percentage of baseline, 10 min after the addition of STC1. * P < 0.05; ** P < 0.001, using t -test. B and C : response of [Ca 2+ ] i to recombinant MCP-1 (100 ng/ml) in the presence or absence of STC1 (100 ng/ml). Panels illustrate Fluo3 fluorescence in representative cells at the times indicated [ B : at time 0 (control) and 15 min after the addition of MCP-1; C : at time 0 (control), 10 min after the addition of STC1, and 15 min after the addition of MCP-1 (which also corresponds to 25 min after the addition of STC1)]. Representative experiment is shown. D : mean [Ca 2+ ] i values at baseline, following the addition of MCP-1, and lastly, following sequential addition of STC1 and MCP-1. The x -axis of the tracings is reported in minutes, whereas the y -axis represents [Ca 2+ ] i (Fluo3 fluorescence ratio). Data shown are representative of 3 independent experiments. * P < 0.01 comparing both values using t -test.
STC1 attenuates MCP-1- and SDF-1 -mediated chemotaxis. Because chemotaxis integrates multiple cellular functions that require calcium signaling, we examined the effect of STC1 on chemokine-mediated cell migration in both the macrophage-like RAW264.7 cells and U937 monoblasts. The effect on chemokinesis (random movement, no chemokine gradient) was also examined (Figs. 2 and 3 ). Treatment of both cell types with STC1 attenuated chemokinesis (Figs. 2 D and 3 C ) as well as MCP-1- (Figs. 2, A and B, and 3 A ) and SDF-1 -mediated (Figs. 2 C and 3 B ) chemotaxis. Denatured STC1 at the same concentrations (10, 50, 100, and 500 ng/ml) was used as a negative control in all assays and showed no effect on chemotaxis or chemokinesis. To avoid repetition, denatured STC1 results are indicated for the MCP-1-mediated chemotaxis assays only (Figs. 2 B and 3 A ) and are not shown for the other assays.
Fig. 2. STC1 inhibits RAW264.7 cell migration. STC1 decreased both chemokine-mediated ( A - C ) and random ( D ) migration of RAW264.7 cells. A : representative micropore membrane appearances with or without STC1 (100 ng/ml) in the upper well of the chemotaxis chamber. Lower chambers contained MCP-1 (200 ng/ml). Fewer cells are trapped in the pores in the presence of STC1 ( right ) indicating decreased migration. B : transfilter cell chemotaxis assays showing the effect of varying doses of STC1 (upper chamber) on MCP-1-mediated chemotaxis. The maximal inhibitory effect was seen with 100 ng/ml of STC1. Denatured (Den.) STC1 had no effect on chemotaxis. C : transwell chemotaxis assays showing inhibitory effect of STC1 on stromal cell-derived factor-1 (SDF-1 )-mediated chemotaxis. Denatured STC1 had no effect (not shown). D : transfilter chemokinesis assays showing inhibitory effect of STC1 on random cell migration. Denatured STC1 had no effect (not shown). All graphs show results expressed as means ± SD ( n = 4). Bonferroni multiple comparison test: ** P < 0.01 vs. no STC1.
Fig. 3. STC1 inhibits U937 cell migration. STC1 decreased both chemokine-mediated ( A and B ) and random ( C ) migration of U937 cells. A : transwell cell chemotaxis assays showing the inhibitory effect of varying doses of STC1 (upper chamber) on MCP-1-mediated chemotaxis. Denatured (Den.) STC1 had no effect on chemotaxis. B : transwell chemotaxis assays showing inhibitory effect of STC1 on SDF-1 -mediated chemotaxis. Denatured STC1 had no effect (not shown). C : transwell chemokinesis assays showing inhibitory effect of STC1 on random cell migration. Denatured STC1 had no effect (not shown). All graphs show results expressed as means ± SD ( n = 4). Bonferroni multiple comparison test: * P < 0.05, ** P < 0.01 vs. no STC1.
In RAW264.7 cells, the inhibitory effect of STC1 appeared particularly dose dependent ( Fig. 2 ) with maximal effect generally achieved at a dose of 100 ng/ml. Figure 2 A shows representative micropore membranes, which illustrate the effect of STC1 (100 ng/ml) on MCP-1-induced RAW264.7 cell migration. In U937 cells, we observed near-maximal inhibition of chemotaxis in the presence of MCP-1 ( Fig. 3 A ) or SDF-1 ( Fig. 3 B ) at a concentration of 10 ng/ml STC1, without an apparent dose-response curve. Furthermore, while chemokinesis was gradually inhibited by STC1 in RAW264.7 and U937 cells (Figs. 2 D and 3 C ), and the half-maximal inhibitory effect was generally observed at/or near 50 ng/ml STC1 in both cell lines, the dose-response curve for inhibition of chemotaxis was different for each cell line. Whereas MCP-1- and SDF-1-mediated chemotaxis was gradually inhibited by STC1 in RAW264.7 cells (half-maximal inhibitory concentration of 50 ng/ml), it was abruptly and almost completely inhibited in U937 cells at the lowest concentration of STC1 (10 ng/ml) used ( Fig. 3, A and B ). Thus the effect of STC1 is not specific for one chemokine and does not appear to be limited to one cell line, suggesting broad effects for STC1 on the immune/inflammatory system.
Upregulation of MCP-1 and STC1 mRNAs in obstructive uropathy. Upregulation of MCP-1 and several chemokines has been shown to follow ureteric obstruction in humans ( 10 ) and experimental animals ( 5, 7, 11 ). In the following experiment, we used ribonuclease protection assay to determine mRNA levels of various inflammatory cytokines in obstructed mouse kidneys and to correlate these with STC1 mRNA levels. As previously reported ( 5, 7, 10, 11 ), ureteric obstruction induces the expression of MCP-1 and a number of chemokines, such as MIP-2, IP-10, and RANTES ( Fig. 4 A ). Similarly, ureteric obstruction increases STC1 mRNA levels in a time-dependent manner with sustained expression through day 21 ( Fig. 4, B and C ). The induction of STC1 mRNA parallels that of MCP-1 in obstructed kidney suggesting a potential functional and regulatory linkage between the two molecules.
Fig. 4. Parallel induction of STC1 and MCP-1 mRNAs in obstructed mouse kidneys. A : 10 µg of total RNA from sham, nonobstructed right (Rt.), or obstructed left (Lt.) kidneys were subjected to ribonuclease protection assay using mcK-5 cDNA template set. A representative gel is shown. Left : labeled probes and they are identified with a connecting line (Ltn, Lymphotactin; RANTES, regulated upon activation, normal T cell expressed and secreted; Eotaxin; MIP, macrophage inflammatory protein; IP-10, interferon-inducible protein-10; TCA-3, T-cell activation protein-3; L32, ribosomal protein L32; GAPDH, glyceraldehyde-3-phosphate dehydrogenase). Right : protected bands corresponding to upregulated mRNAs. B : total RNA (4 µg) was subjected to ribonuclease protection assay using probes specific for STC1, MCP-1, and the ribosomal protein L32. A representative gel is shown. Left : labeled probes. Right : protected bands corresponding to upregulated mRNAs. Arrow points to nonspecific band corresponding to undigested MCP-1 probe. C : densitometric analysis of bands corresponding to kidney STC1 and MCP-1 mRNAs following ureteric obstruction. The x -axis represents mRNA abundance relative to L32, whereas the y -axis represents time following ureteric obstruction. Data show mean values and are representative of 3 independent experiments. ** P < 0.001 relative to time 0; * P < 0.05 relative to time 0; using unpaired t -test.
STC1 is differentially expressed in obstructed rat kidney and localizes to macrophages. To further demonstrate relevance of STC1 to the immune/inflammatory response, it was necessary to show regulated expression of STC1 in a model of renal injury and localization of STC1 protein to inflammatory cells in vivo. Due to the suboptimal performance of available STC1 antibodies for immunohistochemical studies on mouse tissue, we examined rat kidneys for the expression of STC1 at various stages following UUO and sought to determine if STC1 colocalizes with specific macrophage markers. As previously reported, STC1 labeling in the normal kidney was found predominantly in distal nephron segments, whereas weak staining was observed in the proximal tubules ( 6, 17, 26 ). After ureteric obstruction, however, STC1 was strongly upregulated (through day 25 following ureteric obstruction) and was uniformly expressed in all tubular structures as well as in the glomeruli. In addition, STC1 was detected in interstitial cells that did not appear to have direct tubular association ( Fig. 5 ). Dual staining for STC1 and macrophage markers using ED1 clone revealed expression of STC1 in macrophages. Thus contrary to the discrete expression of STC1 in some tubular structures in the normal kidney, STC1 was differentially expressed in obstructed kidney, where it was detected in all tubules, blood vessels, and glomeruli. In addition, STC1 was detected in macrophages, which appeared to have infiltrated the entire kidney. Of note, MCP-1 expression in obstructed kidneys was previously localized to the tubules and macrophages ( 25 ) and thus it parallels the expression of STC1 at these sites.
Fig. 5. STC1 is differentially expressed in obstructed rat kidney and localizes to macrophages. Normal kidney ( A and D ) and tissue from obstructed kidneys { B and E [ day 15 after ureteric (U.) obstruction]; C and F ( day 25 after ureteric obstruction)} were subjected to immunohistochemistry, using anti-hSTC1 ( A - C ), normal rabbit IgG (negative staining control; D ), or ED1 ( B, C, E, and F; please note that B and C represent dual-staining with anti-hSTC1 and ED1, whereas E and F represent single-staining for ED1). Staining for STC1 is shown in red, whereas staining for ED1 is shown in brown; dual-staining for STC1 and ED1 appears as dark maroon (close to black). Arrowheads point to identical cells in the juxtaposed panels ( B vs. E; C vs. F ), showing dual-staining for STC1 and ED1. G, glomeruli; T, tubule. * Collecting duct (based on morphological appearance). A and D : x 140; B, C, E, and F : x 600.
DISCUSSION
These data suggest that STC1, a naturally occurring and ubiquitously expressed calcium-regulating peptide, may be involved in modulating the immune/inflammatory response; it diminishes both spontaneous chemokinesis and the chemotactic response of monocyte cell lines to two different chemokines, MCP-1 and SDF-1. It is also expressed in vivo by macrophages and other cell types in a model of inflammation induced by ureteric obstruction, thus providing indirect evidence that it may have a role in modulating the inflammatory response.
The mechanism by which STC1 inhibits cell movement likely relates to its known effect to inhibit [Ca 2+ ] i. Indeed, we were able to show a dose-response relationship between STC1 and [Ca 2+ ] i in RAW264.7 cells. Similar effects have been shown by us and others ( 19, 30 ) in various cell types, including brain cells and cardiomyocytes. The specific mechanism by which [Ca 2+ ] i is lowered is complex and may involve both alterations in calcium influx across the plasma membrane and effects on intracellular stores. Indeed, in cardiomyocytes we found that STC1 decreases calcium influx through inhibition of L-type channels ( 19 ) and possibly through modulation of IP 3 channels as well (Sheikh-Hamad D, unpublished observations).
Our hypothesis is consistent with the known critical role for [Ca 2+ ] i in monocyte and macrophage function. In macrophages, [Ca 2+ ] i changes have been associated with activation of cellular kinases and phosphatases, degranulation, phagosome-lysosome fusion, cytoskeletal reorganization, transcriptional control, modulation of surface receptors, and regulation of cell adhesion, motility, and chemotaxis ( 16 ), processes that are important for the cellular response to antigenic stimuli. Occupancy of the chemokine receptor by its ligand results in the mobilization of [Ca 2+ ] i through one of three possible pathways [IP 3 receptor pathway, cyclic adenosine diphosphateribose (cADPR)/ryanodine receptor pathway, and S1P pathway]. In the IP 3 receptor pathway, stimulation of G protein-coupled receptors by the chemokine (through G q ) leads to the activation of phospholipase C, which hydrolyzes inositol lipids in the plasma membranes, releasing IP 3 into the cytosol and leaving DAG within the membrane. The primary function of IP 3 is to mobilize calcium from IP 3 -responsive intracellular stores. In the cADPR/ryanodine receptor pathway, nicotin-amide adenine dinucleotide is converted to cADPR through the action of ADP-ribosyl cyclase. cADPR, in turn, stimulates ryanodine receptors in the sarcoplasmic reticulum and induces the release of calcium from this compartment ( 9, 13, 18 ). In the S1P pathway, sphingosine is phosphorylated by sphingosinekinase, generating S1P ( 31 ), which functions to release calcium from endoplasmic reticulum stores in a manner that is independent of IP 3 and/or cADPR. Thus release of calcium from intracellular stores, irrespective of the mechanism employed for this release, plays a major role in macrophage chemotaxis.
The observation in our study that STC1 dose dependently reduced both [Ca 2+ ] i and chemotaxis supports this pathway as the mechanism for the observed effect. Indeed, it is unlikely that STC1 acts by modulating cell membrane receptors for chemokines, as both MCP-1 and SDF act on different receptors (CCR2 and CXCR4, respectively). Furthermore, the effects on chemokinesis suggest a general mechanism, not simply a chemokine-dependent pathway. However, it is important to recognize that the concentration of STC1 needed to maximally inhibit chemokinesis and chemotaxis in RAW264.7 cells ( 100 ng/ml) was equivalent to 20% of the concentration needed for maximal attenuation of [Ca 2+ ] i signals (500 ng/ml; see Fig. 1 ). Thus, while STC1-mediated alterations in [Ca 2+ ] i signals may have an impact on chemokinesis and chemotaxis, we cannot rule out other mechanisms, such as activation/deactivation of intracellular signaling pathways.
Our observations in the two cell lines may be relevant in vivo. Here, we bring evidence for the involvement of STC1 in the response of the kidney to obstructive injury and possibly in the function of macrophages in vivo. In the normal kidney, STC1 is detected in epithelial cells of proximal S 3 -segments and distal nephrons (thick ascending limbs, distal convoluted tubules, and collecting ducts) and may have a role in regulating energy utilization in these metabolically active sites ( 17 ). In advanced stage renal injury that follows long-standing ureteric obstruction, however, in addition to its presence in tubular epithelium, STC1 strongly labels interstitial cells that are not associated with discernible tubular structures. A large number of these cells carry macrophage cell-surface markers, suggesting that STC1 is relevant to macrophage function in response to ureteric obstruction and possibly in other disease states. Furthermore, as the sites of MCP-1 and STC1 expression in obstructed kidneys appear to overlap, at least in macrophages and tubular cells ( 25 ), and show parallel induction at the mRNA level, it is possible that these proteins may function in concert to modulate the immune/inflammatory response in obstructive uropathy.
It is unclear at present whether staining for STC1 in macrophages represents de novo production of STC1 or adsorption of ambient STC1 to binding sites on macrophages. We propose that the role of STC1 in the inflammatory/immune response may relate to the timing and site of its production. Production of STC1 in the injured parenchyma would support a role for this molecule in attenuating the influx of inflammatory cells to the site of injury and thus protect the injured tissue from further influx of cytokines and toxic metabolites. De novo production of STC1 by the inflammatory cells, on the other hand, could potentially play a role in regulating the movement of inflammatory cells and their response to antigenic stimuli.
Through the evolutionary process from fish to mammals, STC1 appears to have maintained functional relevance to calcium homeostasis, as mammalian STC1 has been shown to regulate [Ca 2+ ] i in the normal physiology of the gut ( 15 ), in the adaptive response of brain cells to ischemic injury ( 30 ) and in the regulation of cardiomyocyte L-type channel activity in heart failure ( 19 ). This paper provides another potential function for mammalian STC1; that is, modulation of the inflammatory response in a manner that involves alterations in calcium homeostasis. Indeed, the observation that STC1 inhibits spontaneous movement and the chemotactic response of two monocyte lines to two different chemokines in vitro and the demonstration that it is expressed at inflammatory sites in vivo both argue for a new and unrecognized function for STC1 in disease. Much emphasis has been placed in recent years on factors that drive the inflammatory response, while less is known about the endogenous factors that are involved in blocking or reversing inflammation. We propose that future studies are needed to determine if STC1 may be one of those critical endogenous factors that have intrinsic anti-inflammatory effects to modulate disease.
GRANTS
This work was supported by O'Brien Center Grant (DK-064233-01) and departmental funds furnished to D. Sheikh-Hamad. J. Kanellis is supported by the Australian National Health and Medical Research Council (CJ Martin fellowship) and the Don and Lorraine Jacquot Bequest.
ACKNOWLEDGMENTS
We are deeply grateful to Dr. H. Olsen (Human Genome Sciences, Rockville, MD) for providing rabbit anti-human STC1 antibodies and recombinant human STC1 protein.
【参考文献】
Chang AC, Dunham MA, Jeffrey KJ, and Reddel RR. Molecular cloning and characterization of mouse stanniocalcin cDNA. Mol Cell Endocrinol 124: 185-187, 1996.
Chang AC, Janosi J, Hulsbeek M, de Jong D, Jeffrey KJ, Noble JR, and Reddel RR. A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol Cell Endocrinol 112: 241-247, 1995.
Chen S, Bacon KB, Li L, Garcia GE, Xia Y, Lo D, Thompson DA, Siani MA, Yamamoto T, Harrison JK, and Feng L. In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II. J Exp Med 188: 193-198, 1998.
Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987.
Crisman JM, Richards LL, Valach DP, Franzoni DF, and Diamond JR. Chemokine expression in the obstructed kidney. Exp Nephrol 9: 241-248, 2001.
De Niu P, Olsen HS, Gentz R, and Wagner GF. Immunolocalization of stanniocalcin in human kidney. Mol Cell Endocrinol 137: 155-159, 1998.
Diamond JR, Kees-Folts D, Ding G, Frye JE, and Restrepo NC. Macrophages, monocyte chemoattractant peptide-1, and TGF- 1 in experimental hydronephrosis. Am J Physiol Renal Fluid Electrolyte Physiol 266: F926-F933, 1994.
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.
Galione A. Cyclic ADP-ribose, the ADP-ribosyl cyclase pathway and calcium signalling. Mol Cell Endocrinol 98: 125-131, 1994.
Grandaliano G, Gesualdo L, Bartoli F, Ranieri E, Monno R, Leggio A, Paradies G, Caldarulo E, Infante B, and Schena FP. MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy. Kidney Int 58: 182-192, 2000.
Kaneto H, Fukuzaki A, Ishidoya S, Takeda A, Ogata Y, Sasaki T, Yamada S, and Orikasa S. mRNA expression of chemokines in rat kidneys with ureteral obstruction. Nippon Hinyokika Gakkai Zasshi 91: 69-74, 2000.
Lafeber FP, Flik G, Wendelaar Bonga SE, and Perry SF. Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. Am J Physiol Renal Fluid Electrolyte Physiol 254: F891-F896, 1988.
Lee HC. Cyclic ADP-ribose: a new member of a super family of signaling cyclic nucleotides. Cell Signal 6: 591-600, 1994.
Lewis RS. Calcium signaling mechanisms in T lymphocytes. Annu Rev Immunol 19: 497-521, 2001.
Madsen KL, Tavernini MM, Yachemic C, Mendrick DL, Alfonso PJ, Buergin M, Olsen HS, Antonaccio MJ, Thompson ABR, and Fedorak RN. Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine. Am J Physiol Gastrointest Liver Physiol 274: G96-G102, 1998.
Mandeville JT and Maxfield FR. Calcium and signal transduction in granulocytes. Curr Opin Hematol 3: 63-70, 1996.
McCudden CR, James KA, Hasilo C, and 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.
Putney JW. Excitement about calcium signaling in inexcitable cells. Science 262: 676-678, 1993.
Sheikh-Hamad D, Bick R, Wu GY, Christensen BM, Razeghi P, Poindexter B, Taegtmeyer H, Wamsley A, Padda R, Entman M, Nielsen S, and 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.
Sheikh-Hamad D, Rouse D, and Yang Y. Regulation of stanniocalcin in MDCK cells by hypertonicity and extracellular calcium. Am J Physiol Renal Physiol 278: F417-F424, 2000.
Stannius H. Uber nebenniere bei knochenfischen. Arch Anat Physiol 6: 97-101, 1939.
Tagaki Y, Hirano T, and Yamada J. Effects of the removal of the corpuscles of Stannius on the transport of calcium across the intestine of rainbow trout. Zool Sci 2: 523-530, 1985.
Truong LD, Petrusevska G, Yang G, Gurpinar T, Shappell S, Lechago J, Rouse D, and Suki WN. Cell apoptosis and proliferation in experimental chronic obstructive uropathy. Kidney Int 50: 200-207, 1996.
Truong LD, Sheikh-Hamad D, Chakraborty S, and Suki WN. Cell apoptosis and proliferation in obstructive uropathy. Semin Nephrol 18: 641-651, 1998.
Vielhauer V, Anders HJ, Mack M, Cihak J, Strutz F, Stangassinger M, Luckow B, Grone HJ, and Schlondorff D. Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes. J Am Soc Nephrol 12: 1173-1187, 2001.
Wagner GF, Guiraudon CC, Milliken C, and Copp DH. Immunological and biological evidence for stanniocalcin-like hormone in human kidney. Proc Natl Acad Sci USA 92: 1871-1875, 1995.
Wendelaar Bonga SE and Pang PK. Control of calcium regulating hormones in the vertebrates. Int Rev Cytol 128: 139-213, 1991.
Wu JY, Feng L, Park HT, Havlioglu N, Wen L, Tang H, Bacon KB, Ziang ZH, Zhang XC, and Rao Y. The neuronal repellent slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410: 948-952, 2001.
Zhang J, Alfonso P, Thotakura NR, Su J, Buergin M, Parmelee D, Collins AW, Oelkuct M, Gaffney S, Gentz S, Radman DP, Wagner GF, and 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.
Zhang K, Lindsberg PJ, Tatlisumak T, Kaste M, Olsen HS, and Andersson LC. Stanniocalcin: a molecular guard of neurons during cerebral ischemia. Proc Natl Acad Sci USA 97: 3637-3642, 2000.
Zu Heringdorf DM, Lass H, Alemany R, Laser KTZ, Schmidt MN, Rauen U, Jakobs KJ, and van Koppen CJ. Sphingosine kinase-mediated Ca 2+ signaling by G protein-coupled receptors. EMBO J 17: 2830-2837, 1998.
作者单位：1 The Renal Section, 4 Renal Pathology Laboratory, Department of Pathology, Baylor College of Medicine, 3 Department of Pathology, University of Texas Health Sciences Center, Houston, Texas 77030; and 2 Kidney Laboratory, Austin Research Institute and Department of Nephrology, Austin Hospital, Unive