Cd150 and Cd180 Are Involved in Regulation of Transcription Factors Expression in Chronic Lymphocytic Leukemia Cells

Abstract

Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors. Materials and Methods: Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Results: Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150+) compared to csCD150- CLL cases or normal CD19+ and CD19+CD5+ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150- and csCD150+ CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 and CD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes. Conclusions: Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels.

SLAMF1/CD150 in hematologic malignancies: Silent marker or active player?

“…3). On the other hand, downregulation of IRF4 after CD150 + CD180 ligation also could affect transcriptional programs in CLL B cells [78]. It appears that cell surface expression of CD150 and CD180 is not profitable for CLL B cells.…”

“…CD180 receptor, but not CD40 or BCR could be involved in regulation of CD150 expression in CLL B cells. Ligation of CD180 on CLL B cells leads to increase of CD150 mRNA level that could be explained by CD180-mediated upregulation of EBF1 expression [78]. From the other hand, it was shown that CD40-CD40L interaction alone or together with leukotriene B 4 activates CLL B cells and results in an augmented CD150 cell surface expression [79].…”

CD150 and CD180 are negative regulators of IL-10 expression and secretion in chronic lymphocytic leukemia B cells

“…According to our previous data CD150 or/and CD180 ligation on CLL B cells ex vivo led to a decrease in IRF4 mRNA expression up to 4 times. At the same time, after CD150 or CD180 ligation the BCL6 mRNA expression level was increased compare to control although CLL B cells were characterized by lower basal BCL6 mRNA expression in both CD150and CD150 + subgroups of CLL [8]. Considering the above, it could be hypothesized that CD150 or/and CD180 mediated inhibition of IL-10 expression by CLL B cells is connected with regulation of BCL6 and IRF4` mRNA expression via CD150 and CD180 signaling pathways.…”

“…Our previous data showed that simultaneous activation of CD150 and CD180 cell surface receptors on CLL B cells led to inhibition of main pro-survival Akt and MAPK signaling pathways [7]. Moreover, activation of CD150 and CD180 receptors on CLL B cells led to changes in mRNA expression levels of transcription factors (TFs) that regulate B cells` identity and differentiation (EBF1, IRF4, IRF8, BCL6 and PU.1) [8]. Since both CD150 as adhesion and costimulatory molecule and CD180 as a pattern recognizing receptor could be activated in tumor microenvironment by pattern of common microbial, neo-and autoantigens we tested a hypothesis whether CD150 and CD180-mediated signaling could be directly linked to modification of CLL microenvironment through regulation of cytokines expression and secretion by CLL B cells.…”

“…PBMCs` precipitates obtained from CLL patients were lysed in Triton lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM Tris, pH 8.0. containing cocktails of protease and phosphatase inhibitors (Sigma, USA). Lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis as described earlier [18] The cDNA synthesis and realtime PCR were made by scheme that was reported in previous publications [7,8]. The sequences or primers used in our research are listed in Table 1.…”