Differential signalling during B-cell maturation.

The molecular mechanism by which the antigen receptors (BCR) on B cells can elicit differential maturation state-specific responses is one of the central problems in B-cell differentiation yet to be resolved. Indeed, many of the early signalling events detected following BCR ligation, such as activation of protein tyrosine kinases (PTK), phospholipase C (PLC), phosphoinositide-3-kinase (PI 3K), protein kinase C (PKC) and the RasMAPK (mitogen activating protein kinase) signalling cascades are observed throughout B-cell maturation. However, it is becoming clear that the differential functional responses of these BCR-coupled signals observed during B-cell maturation are dependent on a number of parameters including signal strength and duration, subcellular localisation of the signal, maturation-restricted expression of downstream signalling effector elements/isoforms and modulation of signal by co-receptors. Thus, the combined signature of BCR signalling is likely to dictate the functional response and act as a developmental checkpoint for B-cell maturation.

Harnett MM ，Katz E ，Ford CA 《IMMUNOLOGY LETTERS》

Deficiency of BLNK hampers PLC-gamma2 phosphorylation and Ca2+ influx induced by the pre-B-cell receptor in human pre-B cells.

B-cell linker protein (BLNK) is a component of the B-cell receptor (BCR) as well as of the pre-BCR signalling pathway, and BLNK(-/-) mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. A recent report described the complete loss or drastic reduction of BLNK expression in approximately 50% of human childhood pre-B acute lymphoblastic leukaemias (ALL), therefore we investigated BLNK expression in human pre-B ALL cell lines. One of the four cell lines tested, HPB-NULL cells, was found to lack BLNK expression, and we used these human pre-B ALL cell lines that express and do not express BLNK to investigate the intracellular signalling events following pre-BCR cross-linking. When pre-BCR was cross-linked with anti-micro heavy-chain antibodies, significant phosphorylation of intracellular molecules, including Syk, Shc, ERK MAP kinase, and AKT, and an activation of Ras were observed without regard to deficiency of BLNK expression, suggesting that BLNK is not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signalling. By contrast, phospholipase C-gamma2 (PLC-gamma2) phosphorylation and an increase in intracellular Ca(2+) level mediated by pre-BCR cross-linking were observed only in the BLNK-expressing cells, indicating that BLNK is essential for PLC-gamma2-induced Ca(2+) influx. Human pre-B cell lines expressing and not expressing BLNK should provide an in vitro model for investigation of the role of BLNK in the pre-BCR-mediated signalling mechanism.

Taguchi T ，Kiyokawa N ，Takenouch H ，Matsui J ，Tang WR ，Nakajima H ，Suzuki K ，Shiozawa Y ，Saito M ，Katagiri YU ，Takahashi T ，Karasuyama H ，Matsuo Y ，Okita H ，Fujimoto J ... -  《-》

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

van der Merwe JQ ，Moreau F ，MacNaughton WK 《AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY》

A possible signal transduction pathway for cyclin D2 expression by a pectic polysaccharide from the roots of bupleurum falcatum L. in murine B cell.

Bupleuran 2IIc, a pectic polysaccharide isolated from the roots of bupleurum falcatum L., was previously characterized as a T-cell-independent B cell mitogen. This study focuses on elucidating the mechanism by which bupleuran 2IIc induces cyclin D2 production for inducing mitogenesis in murine B cells. Bupleuran 2IIc was digested with endo-alpha-(1-->4)-D-polygalacturonase and the resulting bupleuran 2IIc/PG-1 ("ramified" region) strongly stimulated cyclin D2 expression. When murine B cells were stimulated with bupleuran 2IIc/PG-1, phosphorylation of tyrosine residues of a number of proteins was observed. Cyclin D2 expression by bupleuran 2IIc/PG-1 was inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A, and the Src family tyrosine kinase inhibitor, PP2, suggesting a possible role for tyrosine kinases. The stimulation by bupleuran 2IIc/PG-1 of cyclin D2 expression was significantly decreased by inhibitors, PI 3-kinase (LY294002 and Wortmannin), PLCgamma (U73122), PKC (H-7), receptor-operated calcium entry inhibitor (SK&F 96365), and calcineurin (FK506). Both PD98059 and U0126, highly selective inhibitors of MEK1 and MEK1/2, respectively, did not strongly suppress the expression of cyclin D2 after stimulation by bupleuran 2IIc/PG-1. The results suggest that (1) bupleuran 2IIc/PG-1 is the active site for induction of cyclin D2 by bupleuran 2IIc, (2) the expression of the cyclin D2 gene by bupleuran 2IIc/PG-1 may be mediated via the activation of PI 3-kinase and PLCgamma followed by activation of PKC and calcium mobilization, and (3) the ERK1/2 cascade is not a central signaling pathway for bupleuran 2IIc/PG-1-induced cyclin D2 expression.

Matsumoto T ，Hosono-Nishiyama K ，Guo YJ ，Ikejima T ，Yamada H ... -  《INTERNATIONAL IMMUNOPHARMACOLOGY》

The signalling profile of recombinant human orexin-2 receptor.

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK(1/2)) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK(1/2) and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK(1/2) and p38 MAPK, with maximal activation at 10 min for ERK(1/2) and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK(1/2) activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK(1/2) activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK(1/2) and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK(1/2) activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.

Tang J ，Chen J ，Ramanjaneya M ，Punn A ，Conner AC ，Randeva HS ... -  《CELLULAR SIGNALLING》