Modulation of the IL-6 Receptor α Underlies GLI2-Mediated Regulation of Ig Secretion in Waldenström Macroglobulinemia Cells

Abstract

Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.

Figure 1. Expression of GLI in IgM secreting cells

(A) Copy number of GLI1, GLI2, and GLI3 in untreated BCWM.1, MWCL-1, and RPCI-WM1 cells determined by qPCR as described in Materials and Methods. Bars represent mean values±S.E. (B) Cells 0.5*10⁶ were fixed/permeabilized and stained with indicated antibody or isotype control followed by FACS analysis. These experiments was repeated 3 times with similar results.

Figure 2. Treatment with GANT61 affects IgM secretion and GLI mRNA expression in WM cells

(A) Relative IgM secretion measured by ELISA for BCWM.1, MWCL-1, and RPCI-WM1 cells treated with 20 µM GANT61 for 48 h when compared to cells treated with only DMSO. (B) mRNA expression of GLI1 and GLI2 in BCWM.1, MWCL-1, and RPCI-WM1 cells treated with 20 µM GANT61 or DMSO for 48 h determined by RT-PCR. (C) Effect of GANT61 (20 µM, 48 h) treatment on relative proliferation of BCWM.1, MWCL-1, and RPCI-WM1 cells when compared with cells treated with DMSO measured by XTT assay. (D) Effect of GANT61 (20 µM, 48 h) treatment on relative viability of BCWM.1, MWCL-1, and RPCI-WM1 cells when compared with control. 0.25 x 10⁶ cells were stained with annexin V and propidium iodide and analyzed by flow cytometry as described in Materials and Methods. (E) Effect of GANT61 (20 μM, 48 h) on relative IgM secretion in BCWM.1 and MWCL-1 cells pretreated with 20 μM Q-VD-OPh (QVD) or DMSO control. Supernatants were used to quantify IgM secretion by ELISA and (F) cells were used to determine cell viability by Annexin V/PI staining. Bars represent mean±S.E. Each experiment was repeated at least 3 times and the bars represent the average of 3-4 pooled biological replicates.

Figure 3. Inhibition of Hedgehog signaling in WM cells has no effect on IgM secretion

(A) mRNA expression of HH signaling components PTCH and SMO in BCWM.1, MWCL-1, and RPCI-WM1 cells determined by RT-PCR. (B) Effect of Cyclopamine (10 µM, 48 h) treatment on IgM secretion measured by ELISA. GLI1 expression was determined by RT-PCR. (C) Effect of Cyclopamine (10 µM, 48 h) treatment on relative proliferation of BCWM.1, MWCL-1, and RPCI-WM1 cells when compared with cells treated with DMSO control by XTT assay. (D) Effect of Cyclopamine (10 µM, 48 h) or DMSO control on relative viability of BCWM.1, MWCL-1, and RPCI-WM1 cells. Cells were analyzed by Annexin V/PI staining. Each experiment was repeated at least 3 times and the bars represent the average of 3-4 pooled biological replicates.

Figure 4. GLI2 inhibition affects expression of IL-6Rα

(A) mRNA expression of cytokine receptors in MWCL-1 cells treated with GANT61 determined by semiquantitative RT-PCR. (B) Relative expression of IL-6Rα in cells treated with GANT61 (20 μM) by qPCR (p=0.0007 for MWCL-1 and p<0.0001 for RPCI-WM1 cells). (C) Relative mRNA expression of IL-6Rα, GLI1 or GLI2 in MWCL-1 and RPCI-WM1 cells transfected with shGLI1 or shGLI2 by qPCR. GAPDH was used as a housekeeping gene. (D) IL-6Rα protein expression in cells transfected with shGLI2 determined by immunoblot. β-actin was used as a loading control. Densitometry on analyzed immunoblot images from pooled 3 independent experiments showing a reduction in IL-6Rα protein expression. (E) 4*10⁶ cells were transfected with either shScr or shGLI2 for 2 days followed by staining for surface IL-6Rα expression by FACS. (F) qPCR for GLI2 expression, IL-6Rα expression and relative IgM secretion in MWCL-1 cells transfected with two different shRNAs targeting GLI2 (shGLI2(1), shGLI2(2). Bars represent mean±S.E. Each experiment was repeated at least 3 times.

Figure 5. GLI2 affects IL-6Rα promoter activity and directly binds to the IL-6Rα promoter

(A) Schematic representing the IL-6Rα promoter with candidate GLI (G) binding sites as determined through bioinformatics analysis. “GLI2” box represents the region where GLI2 binds IL-6Rα and “TSS,” transcription start site. (B) Relative luciferase activity in cells transfected with IL-6Rα promoter-luciferase reporter and treated with either 20 μM GANT61 (+) or DMSO control (−). Cells were harvested after 48 h and changes in luciferase activity were assayed as described in Materials and Methods. (C) Relative luciferase activity in cells transfected with IL-6Rα promoter-luciferase reporter and either scramble shRNA control (Scr) or one of two different shRNA targeting GLI2, shGLI2(1) and shGLI2(2). Cells were lysed 48 h post transfection and used to determine changes in luciferase activity. (D) A chromatin immunoprecipitation (ChIP) assay was performed on cell lysates using antibodies specific for GLI2 or IgG control. qPCR was performed and binding was determined in the IL-6Rα promoter region (−1065/−1294) containing the three candidate GLI binding sites indicated in the schematic above (A). (E) ChIP assay was performed on MWCL-1 cell lysates from cells treated with either 20 uM GANT61 or DMSO for 2 days. qPCR was performed to determine the effect of treatment on GLI2 binding to IL-6Rα promoter. (F) ChIP assay was performed on cell lysates from MWCL-1 cells transfected with either shScr or shGLI2 for 2 days. qPCR was performed to determine the effect of treatment on GLI2 binding to IL-6Rα promoter. .Each experiment was repeated at least 3 times.

Figure 6. IL-6Rα expression rescues GLI2-mediated reduction in IgM secretion

Relative IgM secretion in cells transfected with shGLI2 or Scr and either empty vector or IL-6Rα expression construct (shGLI2 + IL-6Rα). Supernatants were harvested 48 h post-transfection and relative IgM secretion was determined by ELISA. Bars represent mean±S.E of pooled data from 3 independent experiments. Each experiment was repeated at least 3 times.

Figure 7. GLI-mediated regulation of IL-6Rα and IgM secretion in primary IgM secreting mouse B1 cells

(A) Total cells from peritoneal cavities were isolated by intraperitoneal lavage as described in the methods. Cells were treated with 20 μM GANT61 or DMSO control for 2 days followed by staining to determine IL-6Rα expression in B1 cells. Cells were gated on viable lymphocyte population followed by gating to identify B1 cells (CD23^-B220⁺) and B2 cells (CD23⁺B220⁺). IL-6Rα expression was assessed on B1 cells by flow cytometry is shown. Shown is a representative of three independent experiments. (B) Relative IgM secretion from cultures of total peritoneal cells (1*10⁶ cells/ml/well) cultured in triplicate wells in the presence of 20 μM GANT61 or DMSO control for 2 days. Data represents pooled data from 3 independent experiments. (C) Magnetically sorted B1a cells were isolated as described in the methods. Sorted cells (1*10⁶ cells/ml/well) were cultured in triplicate wells in the presence of 20 μM GANT61 or DMSO control for 2 days. Data represents pooled data from 3 independent experiments. (D) Expression of GLI family members by FACS in B1a cells. Magnetically sorted B1a cells were stained for GLI expression as described in the methods followed by FACS analysis. (E) B1a cells were treated with 20 μM GANT61 or DMSO control for 2 days. Cells were harvested and used to determine IL-6Rα expression by qPCR. Data represents pooled data from 3 independent experiments. (F) B1a cells were treated with 20 μM GANT61 or DMSO control for 2 days. Cells were then harvested and used to determine GLI2 and IL-6Rα expression by FACS. Shown is a representative experiment of 3 independent experiments.