Published online before print March 24, 2006
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Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
1Correspondence: Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bldg. 10, Room 11B08, 10 Center Dr., MSC 1876, Bethesda, MD 20892. E-mail: jkehrl{at}niaid.nih.gov
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: B lymphocyte • calcium • chemokine
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most chemoattracant and chemokine receptors couple to the Gi subfamily of heterotrimeric G-proteins [12
]. The binding of ligand activates receptors triggering G
i subunits to exchange guanosine 5'-triphosphate (GTP) for guanosine 5'-diphosphate. This leads to the dissociation of the Gi subunit from its associated Gß
heterodimer, and both components can then activate downstream effectors [13
, 14
]. The release of Gi-associated Gß subunits is essential for triggering directional migration [15
16
17
]. As G subunits possess an intrinsic guanosine triphosphatase (GTPase) activity, GTP hydrolysis leads to the reassembly of heterotrimeric G-protein, causing signaling to cease. B lymphocytes strongly express two members of the Gi subfamily, Gi2 and Gi3 [12
]. Mice deficient in Gnai3 are reportedly without a phenotype [18
]; however, Gnai2–/– mice exhibit defective chemokine receptor signaling as evidenced by impaired lymph node development; poor B cell chemotaxis to CXC chemokine ligand 12 (CXCL12), CXCL13, and CC chemokine ligand 19 (CCL19); and defective homing of transferred B cells to lymph nodes [19
, 20
].
A variety of regulatory mechanisms exists to control the magnitude and duration of heterotrimeric G-protein signaling. One of the most prominent is the existent of GTPase-activating proteins (GAPs), which dramatically accelerate the intrinsic GTPase activity of G subunits [21
22
23
24
25
26
]. These proteins are termed regulators of G-protein signaling (RGS). By reducing the duration that a G subunit remains GTP-bound and thereby, facilitating the reassembly of the inactive, heterotrimeric G-protein, RGS proteins can decrease G-GTP effector activation and decrease free Gß availability. Most of the RGS proteins have GAP activity for Gi and/or Gq subunits. As chemokine receptors use Gi and perhaps Gq to transduce intracellular signals, the presence of a RGS protein in target cells can substantially alter the response to chemokine stimulation. Germinal center B lymphocytes prominently express at least two RGS proteins, RGS1 and RGS13, as do many Burkitt’s lymphoma cells lines [27
, 28
]. Burkitt’s lymphomas likely arise from germinal center B cells and retain many characteristics of these cells. Consistent with a role for RGS1 and RGS13 in regulating the B cell responses to chemokines, the expression of RGS1 or RGS13 in a variety of cell lines reduces CXCR4 and CXCR5 signaling, including impairing increases in intracellular calcium([Ca2+]i), chemotaxis, and extracellular signal-regulated kinase (ERK) activation [27
, 28
]. Conversely, Rgs1 disruption in mice enhances germinal center formation and chemoattractant responses of purified germinal center B cells [29
]. To examine the role of endogenous RGS1 and RGS13 in human B cell lymphoma cell lines, we used a short hairpin (sh)RNA approach to reduce the expression of RGS1, RGS13, or both proteins and explored the effects on chemokine receptor signaling. Our results suggest that RGS1 and RGS13 act together to potently inhibit CXCR4 and to a lesser extent, CXCR5 signaling in these cells and function in the early desensitization of the signaling pathway.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production of constructs for noninducible short hairpin RNAs (shRNAs) and inducible shRNAs
A U6 promoter-based shRNA vector (pENTR/U6, Invitrogen) was used for the noninducible shRNA experiments. Twelve 60 base pair cDNA oligonucleotides were designed and synthesized to target RGS1 or RGS13 mRNA expression. The design of the shRNAs was assisted by the use of web-based software provided by Qiagen Inc. (Valenica, CA; http://www.qiagen.com/Products/GeneSilencing/customSiRna/SiRnaDesigner.aspx) and Invitrogen (http://rnaidesigner.invitrogen.com/rnaiexpress/). Blast searches were performed using the National Center for Biotechnology Information expressed sequence tag database to ensure that the shRNA construct only targeted human RGS1 or RGS13 expression. After annealing, double-strand oligos were inserted to the pENTR/U6 Gateway vector system (Invitrogen). The resulting plasmids were sequenced and then transfected into HeLa cells along with a construct expressing RGS1 or RGS13 fused to GFP. Reduction of target protein levels was analyzed by immunoblotting for GFP expression using a GFP-specific antibody. Those shRNA constructs that showed a 95% reduction of target proteins were selected and transferred to a lentivirus-based system using Gateway protocols (Invitrogen). The final double-strand oligo DNAs for human RGS1 were as follows: top strand, adaptor-stem (sense), loop-stem (antisense) 5'-caccGGAAGTTTCCTAAAGTCTGAAttcaagagaTTCAGACTTTAGGAAACTTCC-3'; bottom strand, adaptor-stem (sense), loop-stem (antisense) 5'-aaaaGGAAGTTTCCTAAAGTCTGAAtctcttgaaTTCAGACTTTAGGAAACTTCC-3'. For human RGS13, they were: top strand, adaptor-stem (sense), loop-stem (antisense) 5'-caccgCTATGCAGTCCAACAACAGttcaagagaCTGTTGTTGGACTGCATAG-3'; bottom strand, adaptor-stem (sense), loop-stem (antisense) 5'-aaaaCTATGCAGTCCAACAACAGtctcttgaaCTGTTGTTGGACTGCATAGc-3'. (Capital letters are target sequences of RGS1 or RGS13.) For negative control, we used empty vectors or firefly (Photinus pyralis) luciferase sequence from pGL3 plasmid (Promega, Madison, WI). The final double-strand oligo DNAs for luciferase shRNA were: top strand, adaptor-stem (sense), loop, 5'-caccgAAGGCTCCTCAGAAACAGCTCttcaagaga; bottom strand, adaptor-loop-stem (antisense) 5'-aaaatctcttgaaGAGCTGTTTCTGAGGAGCCTTc-3'. [Capital letters are sequences from the firefly (P. pyralis) luciferase sequence.] An H1 promoter-based shRNA vector (pENTR/H1/TO, Invitrogen) was used for the inducible shRNA system. This inducible RNA vector contains two copies of regulatory elements from the Escherichia coli Tn10-encoded tetracycline (tet)-resistance operon in the H1 promoter region. In this system, expression of shRNA of interest is repressed by tet repressor protein (tetR) in the absence of tet (or doxycycline) and induced in its presence. The shRNA target sequences for RGS1 and RGS13 were the same as the noninducible system.
Gateway cloning
Gateway recombination cloning reactions were performed as suggested by the manufacturer (Invitrogen). pENTR-U6-shRNA or pENTR/H1/TO-shRNA plasmid was recombined with destination vectors pLenti-Hygro-DEST and pLenti-puro-DEST using the Gateway cloning system to generate the noninducible shRNA constructs (pLenti-Hygro-U6-shRNA and pLenti-puro-U6-shRNA) or inducible shRNA constructs (pLenti-Hygro-H1/TO-shRNA and pLenti-puro-H1/TO-shRNA). To generate the new Gateway DEST vector, the pLenti6/BLOCK-iT-DEST vector was modified. The antibiotic-resistance genes from commercial vectors were amplified by polymerase chain reaction (PCR) using the primers shown in Table 1
. Each resistance module had a 5' PmlI and 3' KpnI site, which were introduced during the PCR. The antibiotic-resistance module were then inserted into pLenti6/BLOCK-iT-DEST vector using PmlI and KpnI sites. Each antibiotic-resistance modules is under control of the simian virus 40 early promoter in the DEST vectors. The entry plasmids, destination plasmids, and final lentiviral shRNA plasmids are shown in Table 2
.
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Table 1. Primers Used for PCR of Resistance Modules
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Table 2. The Entry Plasmids, Destination Plasmids, and Final Lentiviral shRNA Plasmids
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Construction of stable cell lines
To generate inducible cell lines, we made tetR-expressing cell lines using pLent6-T-Rex (pLenti6/TR) virus infections (Invitrogen). The virally infected cells were treated with blastcidin (5–10 µg/ml) for 4 weeks. The blasticidin-resistant cells were selected, and tetR protein level was checked by immunoblotting. Next, we infected shRNA-expressing lentivius into the tetR-expressing cell lines. In this step, the lentiviruses have antibiotic selection markers (hygromycin resistance and/or puromycin resistance), and cells were selected using the appropriate drug. The noninducible cell lines were generated by shRNA expressing viral infection of normal cell lines. The antibiotic selection was conducted for 4 weeks, and stable cell lines were established.
Quantitative reverse transcriptase (RT)-PCR
For the quantitative RT-PCR, a Roche light cycler was used with a Light Cycler Fast Start DNA Master SYBR Green kit (Roche, Indianapolis, IN). Melting curve analysis was performed to control the specificity of PCR product fluorescence. The value of the crossing point was determined for each gene and sample during real-time PCR. The value of the crossing point represents the number of cycles where fluorescence levels of each sample are the same. Plasmids with the appropriate PCR insert subcloned served as the control templates.
HS-Sultan cell migration
Chemotaxis assays were performed using a transwell chamber as described previously [29
]. In some experiments, cells were preincubated with 100 ng/ml pertussis toxin (PTX) for 2 h at 37°C. The cells were washed twice, resuspended in complete RPMI-1640 medium, and added in a volume of 100 µl to the upper wells of a 24-well transwell plate with a 5-µm insert (Corning Glass, Corning, NY). Lower wells contained various doses of chemokines in 600 µl complete RPMI-1640 medium. The number of cells that migrated to the lower well following a 3-h incubation was counted using a flow cytometer. The chemotaxis index was calculated by dividing the number of cells that migrated in response to chemokine by the number of cells that migrated in the absence of chemokine.
Determination of changes in [Ca2+]i
Cells were seeded at a density of 20,000 cells in 100 µl loading medium [RPMI 1640, 10% fetal bovine serum (FBS)] into poly-D-lysine-coated 96-well blackwall, clear-bottom microtiter plates (Nalgene Nunc, Rochester, NY). An equal volume of assay loading buffer (FLIPR Calcium 3 assay kit, Molecular Devices, Sunnyvale, CA) in Hanks’ balanced salt solution supplemented with 20 mM HEPES and 2 mM probenecid was added. Cells were incubated for 1 h at 37°C before adding chemokine, and then the calcium flux peak was measured using a FlexStation (Molecular Devices). The data were analyzed with SOFT max Pro (Molecular Devices). Data are shown as fluorescent counts, and the y-axis was labeled as Lm1.
Immunoblotting
Cell lysates were prepared as described previously [28
]. The detergent-insoluble materials were removed by centrifugation for 10 min at 4°C. Equal amounts of proteins from each sample were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to pure nitrocellulose. Membranes were blocked with 5% bovine serum albumin (BSA) in Tween 20 plus Tris-buffered saline (TTBS) for 1 h and then incubated with an appropriate dilution of the primary antibody in 5% BSA in TTBS for 2 h. The blots were incubated with biotinylated antibody for 1 h and further incubated with streptavidin conjugated to horseradish peroxidase for 1 h. The signal was detected by enhanced chemiluminescence according to the manufacturer’s instruction (Amersham Pharmacia Biotech, Piscataway, NJ). Quantitative changes in the signal were determined following scanning the films using Photoshop (Abode Systems, San Jose, CA).
Imaging
All fluorescent images were collected on an inverted confocal microscope, a Perkin-Elmer Ultraview spinning wheel confocal system (Wellesley, MA) mounted on Zeiss Axiovert 200 and equipped with an argon/krypton laser, an Orca-ERII charged-coupled device camera (Hamamatsu, Japan), and filters suitable for the visualization of fluorescein isothiocyanate and red dyes. The cells were plated on fibronectin-coated glass-bottom culture dishes (MatTek Corp., Ashland, MA) in RPMI with 10% FBS. The cells were maintained in an environment with 5% CO2 at 37°C using an environmental controller system (Carl Zeiss, Germany). Images were recorded over time and collected using a 63x Plan-Aprochromax 1.4 oil immersion objective. To measure [Ca2+]i, individual cells were loaded with a long-wavelength Ca2+ indicator fluo-4/AM (5 µM, Molecular Probes, Eugene, OR), for 1 h at 37°C prior to plating on the culture dishes mentioned above. When excited at 488 nm, fluo-4 exhibits an increase in green fluorescence (525 nm) upon Ca2+ binding. The dye-loaded cells were scanned with the Ultraview confocal, and the fluorescent image was acquired every 15 s followed by a differential interference contrast image. Adobe Photoshop (v. 6.0) was used to prepare composite images and for annotations. Ultraview 5.5 (Perkin-Elmer) and Adobe Photoshop (Adobe Systems) were used for the image processing. The numbers of polarized cells and cells with multiple lamellipodia were determined by visible inspection of 50–100 cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. RGS13 mRNA silencing enhances G-protein-coupled receptor (GPCR) signaling. (A) Selection of a RGS13 shRNA. HeLa cells were transfected with six different shRNA RGS13 (lanes 2–7) constructs and with a construct that expresses RGS13-GFP (lanes 1–7). The level of RGS13-GFP expression was detected by immunoblotting with a GFP-specific antibody. (B) RGS13 mRNA expression in HS-Sultan (HS-S) and an HS-Sultan subline (HS-SR) expressing a RGS13 shRNA or a control shRNA. The level of RGS13 mRNA and actin mRNA was determined by RT-PCR. (C) Decreased RGS13 mRNA expression enhances CXCL12, CXCL13, and PAF-induced increases in [Ca2+]i. HS-Sultan control shRNA (Cont KD) and RGS13 KD cells were stimulated with CXCL12 (100 ng/ml), CXCL13 (1 µg/ml), or PAF (1 µM), and changes in [Ca2+]i were monitored over 3 min. Either duplicate of triplicate determinations is shown. (D) Enhanced migration of RGS13 KD versus control shRNA-expressing cells. Filter-based chemotaxis assays with CXCL12 (100 ng/ml), CXCL13 (1 µg/ml), or PAF (1 µM). Chemotaxis index is shown. The results are from triplicate determinations in two separate experiments.
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Table 3. Quantitative RT-PCR Analysis of Cell Lines
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i activation (data not shown). As the PAF receptor (PAFR) couples to Gi and Gq, and both contribute to the mobilization of [Ca2+]i, the increases in [Ca2+]i in response to PAF were only partially inhibited by PTX (data not shown). Next, we examined the migration of control shRNA and RGS13 shRNA-expressing cells to CXCL12, CXCL13, and PAF using filter-based migration assays. The RGS13 shRNA-expressing cells responded better to each of the chemoattractants than did the control shRNA cells (Fig. 1D) .
Inducible reduction of RGS13 expression also reveals enhanced chemoattractant signaling
The previous experiments compared a cell line expressing a control shRNA to one expressing a RGS13 shRNA. Inherent in the interpretation of these results is the assumption that despite the selection procedures and the long-term expression of two different shRNAs (control and RGS13), the cell lines differ only in their levels of RGS13 expression. An alternative approach, which bypasses the need for selecting separate cell lines expressing a control and the experimental shRNA and allows the comparison of cells shortly after the mRNA KD, is to use an inducible system to express the shRNA. Therefore, we prepared lentivirus, which expresses the shRNA-targeting RGS13 expression under the control of tet operator sequences. We transfected HS-Sultan cells with a pLenti6/TR lentivirus and selected a cell line that stably expressed tetR, referred to it as HS-SultanT. We transduced that cell line with a high-titer lentivirus prepared using pLentis-Hygro-H1/TO-RGS13 shRNA vector (Tables 1
and 2)
and called these cells HS-SultanT13. Next, we induced expression of the RGS13 shRNA by treating the cells with doxycycline for various durations and examined RGS13 expression by RT-PCR. To maximally reduce RGS13 expression required approximately 1 week of treatment. A comparison between the noninduced cells (i.e., control) and cells treated with doxycycline for 1 week is shown (Fig. 2A
). To better quantify the decrease in RGS13 expression, we measured RGS13 mRNA using quantitative RT-PCR and found a 92% reduction in the doxycycline-treated HS-SultanT13 cells (Table 3)
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Figure 2. Inducible silencing of RGS13 mRNA enhances GPCR signaling. (A) RGS13 mRNA in HS-SultanT13 cells induced or not to express the RGS13 shRNA. RT-PCR preformed to assess RGS13 and actin mRNA levels in noninduced (i.e., control) and cells induced to express the RGS13 shRNA (RGS13 KD). (B) Inducible decrease in RGS13 mRNA expression enhances CXCL12, CXCL13, and PAF-triggered increases in [Ca2+]i. HS-SultanT13 cells, induced to express the RGS13 shRNA or not, were stimulated with CXCL12 (100 ng/ml), CXCL13 (1 µg/ml), or PAF (1 µM), and changes in [Ca2+]i were monitored over 3 min. Either duplicate of triplicate determinations is shown. (C) Inducible decrease in RGS13 mRNA expression enhances migration to CXCL12 and PAF. Filter-based chemotaxis assays with CXCL12 (100 ng/ml) or PAF (1 µM). Chemotaxis index is shown. The results are the mean of triplicate determinations for each chemoattractant, which varied less than 10% per individual measurement. (D) Inducible decrease in RGS13 mRNA expression enhances CXCL12-induced ERK activation. Noninduced and induced HS-SultanT13 cells were stimulated with CXCL12 (100 ng/ml) for varying durations. Cell lysates were immunoblotted for pERK and total ERK. Similar results were obtained in two experiments. Fold induction of pERK is shown.
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Reduction of RGS1 expression in HS-Sultan cells minimally enhances chemoattractant signaling
Although HS-Sultan cells express relatively low amounts of RGS1 mRNA, it is readily detectable by RT-PCR. Previous studies had shown that overexpression of RGS1 in HS-Sultan cells blunted responses to CXCL12 and PAF [27
]. To determine whether reducing the basal level of RGS1 expression in HS-Sultan cells augmented chemoattractant signaling, we used a similar approach as we had for the second set of experiments with RGS13. First, we screened a panel of candidate shRNAs targeted at RGS1 expression. We found that three of the six chosen shRNA sequences produced a significant reduction of RGS1-GFP expression and chose one that produced a 90% decrease (Fig. 3A
). We produced the corresponding lentivirus (Tables 1
and 2)
to infect HS-Sultan T cells and selected a stable cell line, HS-SultanT1. A 1-week treatment of that cell line with doxycycline produced an 85% reduction of RGS1 expression (Table 3)
. We compared these cells to noninduced cells (i.e., control) and examined their responsiveness to chemoattractants. We found a modest increase in [Ca2+]i, following chemoattractant exposure compared with the control cells (Fig. 3B)
. We also noted a small increase in specific migration in the doxycycline-treated HS-SultanT1 compared with the noninduced cells (data not shown).
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Figure 3. Inducible silencing of RGS1 mRNA minimally enhances GPCR signaling. (A) Selection of a RGS1 shRNA. HeLa cells were transfected with five different RGS1 shRNA (lanes 1–5) constructs and with a construct that expresses RGS1-GFP (lanes 1–6). The level of RGS1-GFP expression was detected by immunoblotting with a GFP-specific antibody. RGS1 and actin mRNA expression in HS-SultanT1 noninduced cells (lane 7) or RGS1 shRNA-induced cells (lane 8) was determined by RT-PCR. (B) Decreased RGS1 mRNA expression minimally enhances CXCL12, CXCL13, and PAF-induced increases in [Ca2+]i. HS-SultanT1 cells, induced or not, were stimulated with CXCL12 (100 ng/ml), CXCL13 (1 µg/ml), or PAF (1 µM), and changes in [Ca2+]i were monitored over 3 min. Either duplicate of triplicate determinations is shown.
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Figure 4. Reduction of RGS1 and RGS13 expression potently enhances GPCR signaling. (A) Inducible expression of RGS1 and RGS13 shRNAs reduces RGS1 and RGS13 expression. RGS1, RGS13, and actin mRNA expression in HS-SultanT1/13-noninduced (lanes 2, 4, and 6) or doxycycline-induced cells (RGS1/13 KD; lanes 1, 3, 5) determined by RT-PCR. (B) Decreased RGS1 and RGS13 expression enhances PAF-induced increases in [Ca2+]i. HS-SultanT1/13 cells, induced to express the RGS1 and RGS13 shRNAs or not, were stimulated with varying concentrations of PAF, and changes in [Ca2+]i were monitored over 3 min. Each ligand concentration produced a greater increase in [Ca2+]i than the preceding lower concentration. (C) A more pronounced difference between control and double-KD cells at higher PAF concentration. The data from a previous experiment are plotted with percent of maximum [Ca2+]i response on the y-axis versus concentration of PAF on the x-axis. (D) Decreased RGS1 and RGS13 expression enhances a CXCL12-induced increases in [Ca2+]i. HS-SultanT1/13 cells induced to express the RGS1 and RGS13 shRNAs or not were stimulated with varying concentrations of CXCL12, and changes in [Ca2+]i were monitored over 3 min. Each ligand concentration produced a greater increase in [Ca2+]i than the preceding, lower concentration. (E) A more pronounced difference between control and double-KD cells at higher CXCL12 concentrations. The data from a previous experiment are plotted with percent of maximum [Ca2+]i response on the y-axis versus concentration of CXCL12 on the x-axis. (F) Enhanced migration of HS-SultanT1/13-induced cells versus noninduced cells. Filter-based chemotaxis assays with increasing concentrations of CXCL12. Chemotaxis index is shown. The results are from triplicate determinations from two experiments. (G) Enhanced ERK activation in RGS1/13 KD cells. Induced and noninduced HS-SultanT1/13 cells were stimulated with CXCL12 (100 ng/ml) for varying durations. Cell lysates were immunoblotted for pERK and total ERK. Fold increase in pERK levels is shown. Similar results were obtained in three experiments.
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Next, we examined the responsiveness of the induced HS-SultanT1/13 cells to CXCL12-triggered cell migration. The specific migration of the induced cells (RGS1/13 KD) significantly exceeded that of the noninduced cells (Fig. 4F) . Finally, we examined ERK activation in response to stimulation with CXCL12. Treatment with CXCL12 triggered good ERK activation—substantially better than we observed with the noninduced cells (Fig. 4G) .
Decreased RGS1 and RGS13 mRNA expression impairs chemokine receptor desensitization
Next, we tested the ability of the nontreated and doxycycline-treated HS-SultanT1/13 cells to respond to a challenge with a second chemokine after a primary challenge. We exposed the cells to CXCL13 and rechallenged 15 min later with CXCL12 and measured the changes in [Ca2+]i. We found an unimpeded, secondary response to CXCL12 in the noninduced cells and the doxycycline-treated HS-SultanT1/13 cells (Fig. 5A
). However, when we reversed the order, we found that although the induced cells responded normally to the secondary challenge with CXCL13, the noninduced cells did not. This suggested that the primary exposure to CXCL12 had desensitized the noninduced cells to a secondary CXCL13 challenge but had not desensitized the doxycycline-treated HS-SultanT1/31 cells (Fig. 5B)
. As the magnitudes of the responses elicited by the two chemokines differed, we expressed the magnitude of the secondary response as a percentage of that observed in the same cell type to the same chemokine in the absence of a primary challenge (Fig. 5C)
.
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Figure 5. RGS1 and RGS13 mRNA silencing impairs early desensitization of the signaling pathway. (A) CXCL13 does not desensitize CXCL12-induced increases in [Ca2+]i. HS-SultanT1/13 cells, induced to express the RGS1 and RGS13 shRNAs or not, were stimulated with CXCL13 (1 µg/ml) and 15 min later, stimulated with CXCL12 (100 ng/ml). Changes in [Ca2+]i were monitored over 18 min. (B) CXCL12 desensitizes CXCL13-induced increases in [Ca2+]i more so in control than RGS1/13 KD cells. Noninduced and induced HS-SultanT1/13 cells were stimulated with CXCL12 (100 ng/ml) and 15 min later, stimulated with CXCL13 (1 µg/ml). Changes in [Ca2+]i were monitored over 20 min. (C) Analysis of [Ca2+]i tracings in noninduced and induced HS-SultanT1/13 cells (RGS1/13 KD). Areas under each of the calcium tracing shown in A and B are graphed and expressed as a percentage of the expected response for each cell type if it had not been stimulated previously with chemokine. (D) Impaired CXCL12 desensitization in RGS1/13 KD cells compared with control cells. HS-SultanT1/13, induced or not, was stimulated with CXCL12 (25 ng/ml) and 3 (upper left), 5 (upper right), or 15 min (lower) later, restimulated with CXCL12 (100 ng/ml). Changes in [Ca2+]i were monitored over 6, 8, or 20 min as indicated. (E) Analysis of [Ca2+]i tracings in control and RGS1/13 KD cells. Areas under each of the calcium tracing shown in D are graphed. The results are expressed as a percentage of the primary response for the noninduced (Control) and induced cells (RGS1/13 KD).
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Reduction of RGS1 and RGS13 expression alters basal and chemokine-induced cell polarization and the distribution of [Ca2+]i
We also examined the morphology of the nontreated and doxycycline-treated HS-SultanT1/13 cells following exposure to CXCL12. We plated noninduced and induced cells on fibronectin-coated tissue-culture dishes incubated then for 1 h prior to moving the cells to the microscope stage, where they were maintained at 37°C in 5% CO2. The cells were stimulated by adding 25 ng/ml CXCL12 and imaged every 15 s for 5 min and then restimulated 5 min later by adding an additional 100 ng/ml CXCL12 and imaged every 15 s for 5 min. We noted some minor morphological differences between the noninduced and the induced cells even prior to stimulation, as many more induced cells had visible filopodia (Fig. 6A
and 6B
, 0 Minute panels). Five minutes after stimulation, we noted that more of the shRNA-induced cells had a polarized morphology compared with the noninduced cells (Fig . 6A
and 6B
, 5 Minutes panels). By 15 min after the initial stimulation, the majority of the induced cells had polarized or had adopted an unusual morphology of multiple small lamellipodia; in contrast, the noninduced cells had a similar appearance as those 5 min after stimulation (Fig . 6A
and 6B
, note 15 and 20 Minutes panels’ insets). Five minutes after the second stimulation, the noninduced and induced HS-SultanT1/31 cells had not changed noticeably (Fig . 6A
and 6B
, 20 Minutes panels).
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Figure 6. RGS1 and RGS13 mRNA silencing alters cellular morphology. (A and B) Effect of CXCL12 on control and RGS1/13 KD cell morphology. HS-SultanT1/13 cells induced to express the RGS1 and RGS13 shRNAs or not were plated on fibronectin-coated culture dishes, stimulated with CXCL12 (25 ng/ml), and imaged for 5 min at 15 s intervals. Ten minutes later, an additional 100 ng/ml CXCL12 was added, and the cells were imaged for an additional 5 min. Images collected before and 5, 15, and 20 min after the primary stimulation are shown. The 20-min image is 5 min after the secondary stimulus. A 45-µ bar is shown in some of the images. Electronic blow-ups of two RGS1/13 KD cells after stimulation are shown in the insets. (C and D) [Ca2+]i changes in control and RGS1/13 KD cells, respectively. HS-SultanT1/13 cells, induced or not, were loaded with Fluo-4 AM and plated on fibronectin-coated culture dishes prior to addition of CXCL12 (25 ng/ml) to the media. Cells were imaged every 15 s for 3 min. Individual images from the control and RGS1/13 KD cells are shown immediately prior to the addition of chemokine and  130 and 210 s later. The direction of migration of a RGS1/13 KD cell, as assessed by examining its position on sequential images, is indicated by an arrow. (E) Morphological changes in control and RGS1/13 KD cells. The percentage of visibly polarized cells prior to chemokine stimulation, cells with multiple lamellipodia 15 min after exposure to CXCL12, and cells with polarized [Ca2+]i 15 min after exposure to CXCL12 (100 ng/ml). Results from control and induced HS-SultanT1/13 cells (RGS1/13 KD) are from the analysis of 50–100 cells for each condition.
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Germinal center B cells express RGS1 and RGS13 [28 , 29 ]. Among the Burkitt’s lymphoma lines we have tested, all express high levels of RGS13 but variable levels of RGS1. The HS-Sultan cells used in this study constitutively express RGS1, although at relatively low levels compared with activated human B cells and RGS13. HS-Sultan cells and a subline of HS-Sultan cells, which respond poorly to chemokines, proved amendable to the RGS13 shRNA, showing greater than 20-fold reductions in RGS13 mRNA as compared with control shRNA-expressing cells. Furthermore, the placement of RGS13 shRNA under the control of a tet operator allowed the inducible expression of the RGS13 shRNA and the verification of the initial results, which had shown an enhancement in CXCR4, CXCR5, and PAFR-triggered signaling.
Several studies have shown that human germinal center B cells respond poorly to chemoattractants, a result consistent with their expression of RGS1 and RGS13 [29 30 31 32 33 ]. Also, germinal center B cells from Rgs1–/– mice migrate better to CXCL12 than do those from wild-type mice [29 ]. However, a recent study that used germinal center B cells from Bcl-2 transgenic mice observed that their germinal center B cells migrated well to CXCL12 and CXL13 [34 ]. The authors argued that the previously observed refractoriness of germinal center B cells to chemoattractants was caused by their poor in vitro viability, which Bcl-2 expression averted. An alternative explanation may be that the Bcl-2 transgene alters germinal center B cell selection. Two rescue signals, antigen and CD40 ligand, raise Rgs1 and Rgs13 expression levels, respectively [27 , 28 , 35 ]. As the forced Bcl-2 expression allows B cells to survive in the absence of normal rescue signals, the levels of Rgs1 and Rgs13 expression may be low in the Bcl-2 transgenic germinal center B cells.
This study provides little evidence for GPCR-RGS protein selectivity. Although there is some suggestion that RGS13 preferentially regulates CXCR4 signaling, the effect is relatively small, and CXCR5 and PAFR signaling are augmented in the RGS13 single-KD cells. In the double-KD cells, again PAFR, CXCR4, and CXCR5 signaling are all significantly augmented. Thus, the constitutive expression of RGS1 or RGS13 in lymphocytes is likely to impact signaling through multiple GPCRs. Also, the significant enhancement of signaling in the double-KD cells compared with the single-KD indicates that both proteins are contributing to the overall attenuation of the signaling pathway.
In Saccharomyces cerevisiae, the RGS protein Sst2p is critical for desensitization of pheromone-mediated signaling [36 , 37 ]. However, yeast lacks GPCR kinases (GRKs). Mammalian cells likely use RGS proteins (refs. [29 , 38 39 40 41 42 ] and this study) and GRKs to desensitize GPCR-triggered signaling pathways. GRKs function at the level of the receptor by phosphorylating GPCRs. This leads to ß-arrestin recruitment, which uncouples the receptors from G-proteins [43 , 44 ]. A comparison of wild-type and GRK2+/– T cells stimulated with the chemokine CCL4 revealed that the GRK2+/– T cells had a 40% increase in chemotaxis, an enhanced calcium response, and an increase in ERK activation [45 ]. However, it is surprising that the Grk2+/– T cells became as refractory to CCL4 restimulation as the wild-type T cells, demonstrating no impairment in desensitization [45 ]. Unfortunately, the consequences of a complete GRK2 deficiency are unknown as a result of embryonic lethality. The relative importance of RGS proteins and GRKs in GPCR desensitization remains unresolved, in part, because of multiple family members. Our results with B cells suggest that the coreduction of RGS1 and RGS13 impairs the early desensitization of the CXCR4 signaling pathway, and other mechanisms are more important later.
A previous study of B cells from Rgs1–/– mice [20
, 29
] and this study indicate that the absence of RGS1 or RGS13 does not alter the threshold at which a response occurs, i.e., does not shift the dose-response curve to the left, but rather affects the magnitude of the response. As the reduction of RGS1 and RGS13 expression more dramatically enhances chemokine receptor signaling at high-ligand concentrations, the strength of the initial signal may impact RGS1 and/or RGS13 function. We are currently testing whether RGS1 or RGS13 phosphorylation enhances their recruitment and/or activity. An obvious candidate to mediate the phosphorylation would be the GRK proteins, thereby tying together two different mechanisms that desensitize the signaling pathway: one aimed at receptor G-protein coupling and the other at the G subunit. Several alternative mechanisms are also possible. One is a signaling-induced modification of an adaptor protein that recruits and/or activates RGS1 and/or RGS13, such as has been described for RGS9 [46
]. Another possibility is a reciprocal regulation of RGS proteins by phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and Ca2+/calmodulin, whereby PI(3,4,5)P3 binds to RGS proteins to inhibit their GAP activity, which can be reversed by the binding of Ca2+/calmodulin [47
, 48
].
An interesting finding in our analysis of the RGS1/13 KD cells was their propensity to polarize, even in the absence of chemokine stimulation, and their enhanced polarization following stimulation with CXCL12. Their enhanced basal polarization may be accounted for by the presence of GPCR ligands in the serum, including sphingosine 1-phosphate and lysophosphatic acid. Supporting the enhanced propensity of the RGS1 and RGS13 double-KD cells to polarize was the distribution of [Ca2+]i in individual cells. [Ca2+]i in migrating leukocytes is known to be low at the leading edge and high in the rear [49 ], although to our knowledge, the distribution of [Ca2+]i in migrating B lymphocytes has not been reported previously. The finding that the RGS1 and RGS13 double-KD cells develop numerous small lamellipodia is also intriguing. Normally, lymphocytes and other motile cells emit a variable number of lamella, but only a single, dominant one contacts the substrate, expands, and directs cell migration. Although some of the chemokine-stimulated RGS1 and RGS13 double-KD cells had a clearly defined, leading edge and uropod, other cells exhibited multiple small lamella without an obvious dominant one. Thus, one of the functions of RGS proteins in lymphocyte migration may be to help stabilize the primary lamellipodia.
In conclusion, the analyses of a human B cell lymphoma cell line in which RGS1 and RGS13 expression has been silenced provide several useful insights and an impetus for examining whether the strength of the initial activation signal shapes subsequent RGS1 and RGS13 protein function by modifying their location or functional activity. Additional studies should help delineate the relative importance of GRKs and RGS proteins in regulating the signaling output from chemokine receptors and determine their relative importance in desensitizing the signaling pathway.
Received November 28, 2005; revised January 15, 2006; accepted February 14, 2006.
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subunit of Gi, not by activating Gq or Gs Proc. Natl. Acad. Sci. USA 94,14489-14494 dimers released by activation of Gi-coupled receptors Proc. Natl. Acad. Sci. USA 94,14495-14499i is not required for chemotaxis mediated by Gi-coupled receptors J. Biol. Chem. 274,2824-2828 i2-deficient mice Nat. Genet. 10,143-150[Medline] i3, is a member of a protein family with a highly conserved core domain Proc. Natl. Acad. Sci. USA 92,11916-11920 subunits Cell 86,445-452[CrossRef][Medline]-subunits Nature 383,172-175[CrossRef][Medline] i GTPase activity Nature 383,175-177[CrossRef][Medline] signaling responses of B lymphocytes J. Immunol. 164,1829-1838This article has been cited by other articles:
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