1. HGAL, a germinal center specific protein, decreases lymphoma cell motility by modulation of the RhoA signaling pathway
by Xiaoyu Jiang, Xiaoqing Lu, George McNamara, Xiaofei Liu, Elena Cubedo, Kristopher A. Sarosiek, Isidro Sánchez-García, David M. Helfman, and Izidore S. Lossos
Blood
Volume 116(24):
December 9, 2010
©2010 by American Society of Hematology

2. HGAL induces RhoA activation.
HGAL induces RhoA activation. (A) Raji and VAL lymphoma cells, transfected with siRNA for HGAL or scrambled control siRNA 48 hours before the experiments, and HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-mock plasmids, were starved for 8 hours and then seeded on fibronectin for 60 minutes. Cellular extracts were prepared and RhoA, CDC42 and Rac1 pull down assays were performed. HGAL knockdown and equal loading were confirmed by immunoblotting with HGAL and actin antibodies. Results are representative of 3 independent experiments. Densitometry analysis of normalized RhoA-GTP to total RhoA is presented. The values in the control samples were arbitrarily defined as 1. Error bars represent SD; * indicates a statistically significant difference (P < .05) between experimental conditions. Normalized densitometry measurements for CDC42 and HGAL are also shown below the corresponding blots. (B) RhoA pull down assays in CD77+ GC B cells and CD77– cells enriched from normal tonsils. (C) B lymphocytes, enriched from peripheral blood of healthy volunteers, were transfected with control or HGAL-encoding plasmids. 48 hours later the cells were stimulated with LPA and RhoA activity was assessed in triplicates by G-Lisa RhoA Activation Assay kit (Cytoskeleton Inc). * indicates a statistically significant difference (P = 3.3 × 10−5). Fraction of the same lymphocytes was used in IL-6 chemotaxis assay, demonstrating significant (P = 6.6 × 10−4) inhibition in chemotaxis of HGAL-expressing normal lymphocytes.
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

3. HGAL induces activation of RhoA downstream effectors regulating actin cytoskeleton.
HGAL induces activation of RhoA downstream effectors regulating actin cytoskeleton. (A-B) Raji and VAL lymphoma cells, transfected with siRNA for HGAL or control siRNA, and HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-plasmids, were starved for 8 hours and then seeded on fibronectin for 60 minutes. (A) ROCK enzymatic activity was measured in triplicates in the indicated cells. Data are presented as mean ± SD of the mean. (B) Cellular lysates were immunoblotted for indicated proteins. Densitometry measurements of respective phosphorylated to nonphosphorylated proteins are presented. The values in control specimens were arbitrarily defined as 1. (C) Raji lymphoma cells, transfected with siRNA for HGAL or control siRNA were starved for 8 hours and then seeded on fibronectin for 90 minutes. The cells were stained with anti-HGAL (red), anti-pTyr (green) and DAPI (blue). Interference reflection contrast (IRM) images were obtained and p-Tyr staining intensity was measured and depicted as mean ± SD of the mean. (D) VAL or Raji cells transfected with siRNA for HGAL or control siRNA were starved for 8 hours and then left unstimulated or treated with LPA (1.0 μg/mL) for 45 seconds followed by staining with Alexa-488 phalloidin and analyzed by flow cytomety. (E-F) Serum-starved HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-plasmids were seeded on FN-coated slides for 90 minutes. (E) The cells were stained with anti-pTyr (green), anti-paxillin (red) and DAPI (blue); and (F) rhodamine-labeled phalloidin (red); IRM images were obtained and visualized on a Carl Zeiss LSM510/UV confocal microscope. The intensity of each staining was calculated in 25 cell tetrads as described in supplemental Mehtods and depicted as mean ± SD of the mean for p-Tyr and paxillin (E). Results in panels A through E are representative of 3 independent experiments. * indicate statistically significant difference (all below P < .01).
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

4. HGAL augments transcriptional effects and transformation potential of RhoA.
HGAL augments transcriptional effects and transformation potential of RhoA. (A) Raji lymphoma cells were transfected with SRF-driven luciferase reporter construct pSRE-Luc and pRL-TK plasmids and with either siRNA for HGAL or scrambled control siRNA. Forty-eight hours after transfection the cells were starved for 8 hours and then either plated on FN-coated or noncoated plates for 60 minutes followed by determination of luciferase activity. Numbers refer to luciferase activities representing means + SD of the mean of 3 independent experiments, each performed in triplicate. * indicate statistically significant (all below P < .01) difference. (B) HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-mock plasmids were transiently transfected with pSRE-Luc and pRL-TK. Forty-eight hours after transfection the cells were starved for 8 hours and then either plated on FN-coated or noncoated plates for 60 minutes and luciferase activity was determined as described in panel A. (C-D) NIH 3T3 cells were transfected with pcDNA3.1-mock, or pcDNA3.1-RhoAQ63L, pcDNA3.1-HGAL and pcDNA3.1-PDZ-RhoGEF alone or together with pcDNA3.1-HGAL (C) or pcDNA3.1-HGAL with control or RhoA siRNA (D). Cells were stained 3 weeks after transfection. Representative plates are depicted. Foci of transformation were counted in triplicate plates in independent transfections and the number of foci per μg of transfected DNA for each condition is shown as mean ± SD of the mean. * indicate statistically significant difference (all below P < .001 in panel C and P = .016 in panel D).
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

5. HGAL interacts with PDZ-RhoGEF and LARG and stimulates guanidine exchange activity.
HGAL interacts with PDZ-RhoGEF and LARG and stimulates guanidine exchange activity. (A) Cellular lysates were extracted from unmanipulated isolated GC B lymphocytes and HeLa cells stably transfected with pcDNA3.1-HGAL, Raji and VAL cells seeded on fibronectin and subjected to immunoprecipitation with anti–PDZ-RhoGEF and LARG or HGAL antibodies, as well as control antibodies, followed by anti-HGAL and anti–PDZ-RhoGEF or anti-LARG Western immunoblotting, respectively. (B-C) Raji (B) and HeLa cells stably transfected with pcDNA3.1-HGAL (C) were seeded on fibronectin for 90 minutes and stained with DAPI (blue nuclear staining), antibodies to HGAL (green) and PDZ-RhoGEF or LARG (red). Slides were viewed on a Carl Zeiss LSM510/UV confocal microscope with 63×/1.4 NA plan apochromat objective lens and 2× zoom and processed with Zeiss LSM510 AIM 3.2 SP2 confocal microscope software. Z stack linescan images demonstrating proteins colocalization at the cell membrane (arrow) are shown; higher resolution images are shown in supplemental Figure 6. (D) PDZ-RhoGEF was immunoprecipitated from Raji cells and used in the RhoA N-methylanthraniloyl exchange assay, either alone or with purified HGAL protein. Purified Dbs protein served as a positive control, while immunoprecipitates with beads only, purified HGAL alone and water were used as negative controls. Results are representative of 3 independent experiments.
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

6. PDZ-RhoGEF interacts with HGAL through DH domain.
PDZ-RhoGEF interacts with HGAL through DH domain. (A) Structure of wild-type and 6 different V5 epitope tagged truncated mutants of PDZ-RhoGEF that were used to transfect Raji lymphoma cells expressing endogenous HGAL (B) and HEK-293T cells cotransfected with pcDNA3.1-HGAL vector (C). PDZ: PDZ domain; RGS: regulators of G protein signaling domain, B: protein sequence binding to actin, DH: Dbl-homology domain, PH: pleckstrin-homology domain. (B-C) Total cellular extracts prepared from the transfected Raji (B) and HEK-293T (C) cells were immunoprecipitated with anti-HGAL and anti-V5 antibodies and analyzed by Western blotting with anti-V5 and anti-HGAL antibodies, as indicated. The corresponding total cellular lysates were subjected to Western blot analysis using anti-HGAL and anti-V5 antibodies. (D) The purified GST-( )PDZ-RhoGEF or GST proteins were incubated with purified TRX-HGAL or TRX proteins for 12 hours. The co-precipitated HGAL and ( )PDZ-RhoGEF were detected by immunoprecipitation with anti-HGAL and anti-GST antibodies followed by Western blotting with anti-GST and anti-HGAL antibodies, as indicated. Results are representative of 3 independent experiments.
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

7. HGAL interacts with PDZ-RhoGEF through its C-terminal PDZ binding motif.
HGAL interacts with PDZ-RhoGEF through its C-terminal PDZ binding motif. (A) HeLa cells were co-transfected with pcDNA3.1-PDZ-RhoGEF and one of the following plasmids: pcDNA3.1, pcDNA3.1-HGAL, pcDNA3.1-HGALΔPDZ and pcDNA3.1-(1-118)HGAL. Total cellular lysates were prepared and immunoprecipitated with anti-HGAL and anti–PDZ-RhoGEF antibodies and analyzed by Western blotting with anti–PDZ-RhoGEF and anti-HGAL antibodies, as indicated. The corresponding total cellular lysates were subjected to Western blot analysis using anti-HGAL and anti–PDZ-RhoGEF antibodies. (B-C) HeLa cells transfected with pcDNA3.1, pcDNA3.1-HGAL or pcDNA3.1-HGALΔPDZ were used for RhoA pull-down (B) and analysis of luciferase activity from the SRF-driven luciferase reporter construct pSRE-Luc as described in “Luciferase reporter assays” and shown in Figures 1B and 3A. Numbers refer to luciferase activities representing means + SD of the mean of 3 independent experiments, each performed in triplicate. * indicate statistically significant difference (all below P < .01). Results in panels A and B are also representative of 3 independent experiments.
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology

8. Cooperative effects of HGAL, PDZ-RhoGEF and LARG on RhoA activation and lymphocyte motility.
Cooperative effects of HGAL, PDZ-RhoGEF and LARG on RhoA activation and lymphocyte motility. (A) Raji cells transfected with either control siRNA or siRNA for HGAL alone or in combination with siRNAs for PDZ-RhoGEF, LARG or both were seeded on fibronectin. Cellular lysates were prepared and used for RhoA pull-down assay and Western blotting with anti-HGAL, PDZ-RhoGEF, LARG and actin antibodies. Densitometry analysis of normalized RhoA-GTP to total RhoA from 3 independent Western blot experiments is presented. The values in the control specimens were arbitrarily defined as 1. Error bars represent SD. (B) HeLa cells stably transfected with pcDNA3.1 or pcDNA3.1-HGAL were transfected with siRNAs for PDZ-RhoGEF, LARG or both and were seeded on fibronectin. Cellular lysates were prepared and used for RhoA pull-down assay and Western blotting with anti-HGAL, PDZ-RhoGEF, LARG and actin antibodies. Densitometry analysis of normalized RhoA-GTP to total RhoA from 3 independent Western blot experiments is presented. The values in the control specimens were arbitrarily defined as 1. Error bars represent SD. (C) VAL lymphoma cells were transfected with control siRNA or siRNA for HGAL and siRNAs for PDZ-RhoGEF, LARG or both. 48 hours after siRNA transfection, the cells were used for IL-6 and SDF1 chemotaxis assay performed in triplicate, as described in the “Chemotaxis assays.” Means + SD of the mean of 3 independent experiments are demonstrated. Statistically significant differences with *P < .05 and **P < .01.
Xiaoyu Jiang et al. Blood 2010;116:
©2010 by American Society of Hematology