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Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways.

Anbazhagan K, Rabbind Singh A, Isabelle P, Stella I, Céline AD, Bissac E, Bertrand B, Rémy N, Naomi T, Vincent F, Rochette J, Lassoued K - Immun Inflamm Dis (2013)

Bottom Line: Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation.PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways.Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

View Article: PubMed Central - PubMed

Affiliation: Inserm/UMR925, Université Picardie Jules Verne, Laboratoire d'Immunologie, UFR de Médecine 3, rue des Louvels, 80036, Amiens, France.

ABSTRACT
Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

No MeSH data available.


Related in: MedlinePlus

Ordering of SRC, SYK, AKT and MAPK in pre-BCR signalling cascade. Pre-BCR was cross-linked using anti-µHC F(ab')2 antibody in presence or absence of BAY 6136-06 (SYK inhibitor) (A), Ly294002 (PI3K/AKT inhibitor) (B), U0126 (MEK1/2 inhibitor) (C), PP2 (SRC inhibitor) or PP3 (inactive homolog of PP2) (D). Total cell lysates were analysed by western blotting for p-BLNK, p-AKT, p-ERK, p-GSK3β or p-SYK. The blots were re-probed for non-phosphorylated forms of all signalling molecules and actin as loading control. Data represented is one of two independent experiments. Med: medium only (RPMI 1640).
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fig01: Ordering of SRC, SYK, AKT and MAPK in pre-BCR signalling cascade. Pre-BCR was cross-linked using anti-µHC F(ab')2 antibody in presence or absence of BAY 6136-06 (SYK inhibitor) (A), Ly294002 (PI3K/AKT inhibitor) (B), U0126 (MEK1/2 inhibitor) (C), PP2 (SRC inhibitor) or PP3 (inactive homolog of PP2) (D). Total cell lysates were analysed by western blotting for p-BLNK, p-AKT, p-ERK, p-GSK3β or p-SYK. The blots were re-probed for non-phosphorylated forms of all signalling molecules and actin as loading control. Data represented is one of two independent experiments. Med: medium only (RPMI 1640).

Mentions: In order to determine the roles of SYK, PI3K and MAPK in pre-BCR signalling, cells were treated with their respective pharmacological inhibitors followed by stimulation with anti-μ F(ab')2 antibodies. Use of BAY61-3606 (SYK inhibitor) resulted in complete inhibition of pre-BCR-induced BLNK, AKT and ERK1/2 phosphorylation (Fig. 1A), p105 NF-κB1 degradation (Fig. 2A and C) and subsequent abrogation of cell growth (not shown). LY294002 (PI3K inhibitor) prevented AKT and GSK3β phosphorylation, but did not modify ERK1/2 phosphorylation (Fig. 1B). PI3K inhibition also prevented p105 NF-κB1 degradation indicating that pre-BCR-mediated NF-κB activation is PI3K-dependent (Fig. 2D). In contrast, treatment of pre-B cells with the U0126 (MEK1/2 inhibitor) resulted in the enhancement of the pre-BCR-induced NF-κB activation. This is shown by an earlier and enhanced degradation of p105 NF-κB without affecting AKT phosphorylation, indicating that negative role is played by the MAPK pathway in preventing NF-κB activation (Figs. 2E and 1C). Consequences of AKT or ERK knock-down by means of shRNA could not be analysed since electroporation of their respective or control shRNA plasmids resulted in high cell mortality.


Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways.

Anbazhagan K, Rabbind Singh A, Isabelle P, Stella I, Céline AD, Bissac E, Bertrand B, Rémy N, Naomi T, Vincent F, Rochette J, Lassoued K - Immun Inflamm Dis (2013)

Ordering of SRC, SYK, AKT and MAPK in pre-BCR signalling cascade. Pre-BCR was cross-linked using anti-µHC F(ab')2 antibody in presence or absence of BAY 6136-06 (SYK inhibitor) (A), Ly294002 (PI3K/AKT inhibitor) (B), U0126 (MEK1/2 inhibitor) (C), PP2 (SRC inhibitor) or PP3 (inactive homolog of PP2) (D). Total cell lysates were analysed by western blotting for p-BLNK, p-AKT, p-ERK, p-GSK3β or p-SYK. The blots were re-probed for non-phosphorylated forms of all signalling molecules and actin as loading control. Data represented is one of two independent experiments. Med: medium only (RPMI 1640).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217539&req=5

fig01: Ordering of SRC, SYK, AKT and MAPK in pre-BCR signalling cascade. Pre-BCR was cross-linked using anti-µHC F(ab')2 antibody in presence or absence of BAY 6136-06 (SYK inhibitor) (A), Ly294002 (PI3K/AKT inhibitor) (B), U0126 (MEK1/2 inhibitor) (C), PP2 (SRC inhibitor) or PP3 (inactive homolog of PP2) (D). Total cell lysates were analysed by western blotting for p-BLNK, p-AKT, p-ERK, p-GSK3β or p-SYK. The blots were re-probed for non-phosphorylated forms of all signalling molecules and actin as loading control. Data represented is one of two independent experiments. Med: medium only (RPMI 1640).
Mentions: In order to determine the roles of SYK, PI3K and MAPK in pre-BCR signalling, cells were treated with their respective pharmacological inhibitors followed by stimulation with anti-μ F(ab')2 antibodies. Use of BAY61-3606 (SYK inhibitor) resulted in complete inhibition of pre-BCR-induced BLNK, AKT and ERK1/2 phosphorylation (Fig. 1A), p105 NF-κB1 degradation (Fig. 2A and C) and subsequent abrogation of cell growth (not shown). LY294002 (PI3K inhibitor) prevented AKT and GSK3β phosphorylation, but did not modify ERK1/2 phosphorylation (Fig. 1B). PI3K inhibition also prevented p105 NF-κB1 degradation indicating that pre-BCR-mediated NF-κB activation is PI3K-dependent (Fig. 2D). In contrast, treatment of pre-B cells with the U0126 (MEK1/2 inhibitor) resulted in the enhancement of the pre-BCR-induced NF-κB activation. This is shown by an earlier and enhanced degradation of p105 NF-κB without affecting AKT phosphorylation, indicating that negative role is played by the MAPK pathway in preventing NF-κB activation (Figs. 2E and 1C). Consequences of AKT or ERK knock-down by means of shRNA could not be analysed since electroporation of their respective or control shRNA plasmids resulted in high cell mortality.

Bottom Line: Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation.PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways.Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

View Article: PubMed Central - PubMed

Affiliation: Inserm/UMR925, Université Picardie Jules Verne, Laboratoire d'Immunologie, UFR de Médecine 3, rue des Louvels, 80036, Amiens, France.

ABSTRACT
Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

No MeSH data available.


Related in: MedlinePlus