Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma

Abstract

Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow. This microenvironment facilitates crucial interactions between the cancer cells and stromal cell types that permit the tumour to survive and proliferate. There is increasing evidence that the bone marrow mesenchymal stem cell (BMMSC) is stably altered in patients with MM-a phenotype also postulated to exist in patients with monoclonal gammopathy of undetermined significance (MGUS) a benign condition that precedes MM. In this study, we describe a mechanism by which increased expression of peptidyl arginine deiminase 2 (PADI2) by BMMSCs in patients with MGUS and MM directly alters malignant plasma cell phenotype. We identify PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through its enzymatic deimination of histone H3 arginine 26, PADI2 activity directly induces the upregulation of interleukin-6 expression. This leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26.

Figure 1

Heatmap of transcriptomic analysis from 30 BMMSC samples, 10 each from control, MGUS and MM patients shows that cells from patients with MGUS and MM cluster together, and are different from those isolated from healthy individuals. One hundred and eighty seven genes were identified with a cutoff for gene expression change of greater than 1.5-fold, and P<0.001.

Figure 2

(a) Three of the top five upregulated transcripts that differed between control and ‘disease' (MGUS/MM) BMMSCs identified from the transcriptomic analysis were the peptidyl arginine deiminase, PADI2 (a), filaggrin (b) and the urea transporter, SLC14A1 (c). (d) PADI2 mRNA expression in BMMSCs from controls, MGUS and MM patients compared with beta-actin, analysed using Taqman, plotted on a logarithmic scale. Significant changes shown were calculated using Mann–Whitney tests. *P<0.05, **P<0.005, ****P<0.0001. (e) BMMSCs derived from patients with MGUS and MM have greatly increased expression of PADI2 and citrullination of histone H3 on residue R26 (R26cit) compared with healthy controls. Citrullination of other arginine residues on histone H3 (R2,8,17) under the control of PADI4 are not similarly consistently increased. ‘C_L' and ‘C_H' indicate control samples (C_L: low expressing, C_H: high expressing) analysed on each blot in order for blots to be more easily compared.

Figure 3

(a) Expression of PADI2 protects plasma cells from chemotherapy-induced apoptosis. JJN3 cell apoptosis as a percentage of total cells, either untreated (‘JJN3'), or 24 h after treatment with 10 nm bortezomib (‘+BTM'). When co-cultured with either a low PADI2-expressing (‘PADI2 low') or high PADI2-expressing (‘PADI2 High') BMMSCs across a Boyden chamber, JJN3 cells are partially protected from bortezomib-induced apoptosis, with extent of protection correlating with PADI2 expression. Two primary BMMSC lines were used per assay, each repeated n=3. Data were analysed by Kruskal–Wallis test (P<0.0001), with Dunn's multiple comparisons test to test for those values that were significantly different. Error bars show s.d. (b) Inhibition of total PADI activity in the PADI2 High expressing lines using the pharmacological inhibitor, Cl-Amidine (‘CL') significantly reduces acquired protection from bortezomib. Data were analysed using Mann–Whitney test, error bars show s.d. **P=0.002 (c) IL-6 expression is significantly increased in BMMSCs derived from patients with MGUS and MM compared with controls. IL-6 mRNA expression was normalised to beta-actin. Significant changes shown between groups were calculated using Mann–Whitney tests. ****P<0.0001. Error bars indicate 10th-90th percentile (d) Bone marrow concentrations of IL-6 are also increased in patients with MGUS and MM. Mann–Whitney tests were again used to calculate significance of differences between groups. ***P=0.0005, ****P<0.0001. (e) Scatterplot of the expression of PADI2 against IL-6 as measured in BMMSCs from 38 patients with MM and MGUS shows that their expression is highly correlated (r²=0.540, non-linear fit).

Figure 4

(a) Confirmation of knockdown by siPADI2 by two independent siRNA sequences. (b) The expression of the BMMSC component of critical chemokine signalling axes in myeloma (IL-6, CXCL12 and cMET) is modulated by PADI2 expression in the immortalised BMMSC cell line, HS5. Expression of all three transcripts are reduced in response to reduction in PADI2 expression (siPADI2), or the addition of the pan-PADI inhibitor, Cl-Amidine (200 μm, 72 h). However, siPADI4 does not alter expression of any of the transcripts. ** and *** indicate the level of significance assigned by a Dunn's comparison post-test after a significant ANOVA for each dataset (P<0.0001 [IL-6] P=0.0002 [CXCL12] P=0.0019 [cMET]). (c) IL-6 expression, as measured through its secretion into the tissue culture medium by the HS5 cell line, is reduced after treatment with Cl-Amidine and/or siPADI2. Data shown are mean of n=3±s.d. *P=0.039, unpaired student's t-test with Welch's correction. (d) Overexpression of PADI2 increases IL-6 expression, whereas the catalytic mutant, C645S, does not increase expression over basal levels. Data shown are mean of n=3±s.d. **P=0.004, unpaired student's t-test with Welch's correction.

Figure 5

(a) Diagram showing the coverage of the IL-6 promoter region by the primer pairs used in the chromatin immunoprecipitation analysis. Pull-down of chromatin associated with either citrullinated R26 (b, d) or trimethylated K4 (c, e) of histone H3, show that both inhibition of PADI activity by Cl-Amidine (b, c) and reduction of PADI2 expression (through siRNA, d, e) reduce the enrichment of these marks on and downstream of the IL-6 transcription start site, indicating that PADI2 activity is required for full activation of transcription from this locus. For all panels, error bars are ±s.e.m.

Figure 6

Model of how PADI2 activity in BMMSCs directly contributes to malignant plasma cell phenotype in patients with MGUS and MM. PADI2 citrullinates histone H3 at residue R26 within the promoter region of the IL-6 gene, which increases its expression and secretion. IL-6 can then mediates its effects on other cells within the bone marrow, including the malignant plasma cells, which become more resistant to chemotherapeutic agents, such as bortezomib.