The Molecular Mechanism Of SHP2 Actions In IL-6 Inducing The Redifferentiation Of Gc-derived B-NHL

AIMS: The redifferentiation of B lymphocytes depends primarily on alterations in various microenvironments, where B cells reside. IL-6, a cytokine expressed in germinal center (GC), modulates B cell differentiation via its specific receptor and SHP2 activity. The passage of human B cells in the GC has a fundamental function for the establishment of effective humoral immunity. In the GC, antigen-specific B cells alter their immunoglobulin (Ig) gene expression by somatic hypermutation(SHM) and class switch recombination(CSR) inducing intensive proliferation. CSR and SHM both induce double strand DNA breaks resulting in aberrant chromosome translocations that are associated with B cell lymphoma such as non-Hodgkin GC-derived B cell Lymphoma (GC-derived B-NHL). IL-6 enhances the frequency of SHM and CSR increasing susceptibility to malignant lymphoma.IL-6 might regulate the development and differentiation of normal B cells by modulation of the SHP2 activity. SHP2 encoded by PTPN11, a key member of tyrosine phosphatase family proteins (PTPs), is identified as the first PTP proto-oncogene. Notably, one genetic disorder in humans, Noonan Syndrome, has been linked to gain-of-function mutations in the SHP2/PTpN11 gene. SHP2 as a cytoplasmic protein tyrosine phosphatase functions in multiple fundamental cellular signaling pathways (RAS-ERK, PI3K-AKT, JAKs-STAT3) regulating cell proliferation, differentiation, apoptosis, and motility. We have noted that SHP2 plays a critical role in differentiation of B lymphocytes in the literature. In this study, we focus on investigation the role of SHP2 in IL-6 mediated transformation and proliferation of GC-derived B-NHL cells.METHODOLOGY/PRINCIPAL FINDINGS: We transfect either scrambled siRNA or SHP2 siRNA pools into human GC-derived B-NHL cells (Daudi, Raji, Ramos cell lines) to silence the SHP2 gene.①SHP2 knockdown leads to a decrease in Erkl/2 phosphorylation concomitant with a reduction in phosphorylation of tyrosine residue Y416 in the activation loop of Src. Whereas Stat3 and Akt phosphorylations are not affected.②Inhibition of SHP2 expression in cell lines significantly impairs cellular proliferation in comparison to controls. To understand whether the observed low proliferation rate is due to cell cycle arrest, we do cell cycle analysis with propidium iodide staining.③Cell cycle analysis reveals that SHP2 knockdown leads to a significant increase in the percentage of GC-derived B-NHL cells in the G1 phase and a corresponding decrease of cells in the S and G2-M phases, without changes in the apoptotic fraction as indicated by the sub-GO-G1 populations. To verify if the SHP2 PTP activity is required for GC-derived B-NHL cell growth, we transfect a PTP-inactive SHP2 mutant (SHP2RE) in which the catalytic Arg-465 replaced with Glu into GC-derived B-NHL cells.④Similar to SHP2 knockdown in GC-derived B-NHL cell lines, the R/E SHP2 protein also inhibits ERK1/2 and Src phosphorylation as well as cell proliferation.Daudi, Raji and Ramos cells are characterized by the translocation of t(8;14)(q24;q32) of the c-myc into the IG loci and high expression of the GC/centroblast marker CD77 as well as critical GC factors such as Bcl6, AICDA, E2A and Pax5. To investigate how SHP2 may contribute to the expression of GC-like features in B cells, we have assessed the protein levels of the GC factors.⑤Indeed, SHP2 inhibition leads to a decrease in CD77 expression in Daudi, Raji and Ramos cells.⑥Concomitantly, the protein levels of Bcl6, E2A, AICDA and Pax5 are also reduced in these cells.⑦Biochemical analysis shows that SHP2 knockdown results in down-regulation of c-myc in assayed cells.⑦The frequence of the translocation of t(8;14)(q24;q32) is decreased in Raji and Ramos cells as assessed by PCR.⑨We find that Src and Erkl/2 activities are essential for keeping the constitutive GC phenotype in BL cells.In addition, we also investigate the role of SHP2 in IL-6 signaling and examine its activity to induce the differentiation of GC-derived B-NHL cells into plasma cells.⑩Flow cytometry results show that GC-derived B-NHL cells with SHP2 knockdown display more CD138-positive and Western blotting indicates that expressions of Blimp1 and XBP1 are also increased. These results suggest that SHP2 is a key mediator for IL-6 induced redifferentiation of GC-derived B-NHLCONCLUSION: In this study, we demonstrate:1. SHP2 regulates GC-derived B-NHL cells proliferation in vitro.2. Reduction of SHP2 expression leads to a decrease in constitutive GC phenotype in GC-derived B-NHL.3. SHP2 also acts as a key mediator for IL-6 signaling that contributes to differentiation of GC-derived B-NHL cells into plasma cells.