No abstract

Osmoregulation, inhibitory neurotransmission and pH balance depend on chloride ion (Cl-) flux. In intestinal epithelial cells, apical Cl- channels control salt and fluid secretion and are, in turn, regulated by agonists acting through cyclic nucleotides and internal calcium ion concentration ([Ca2+]i). Recently, we found that muscarinic pretreatment prevents [Ca2+]i increases from eliciting Cl- secretion in T84 colonic epithelial cells. By studying concomitant inositol phosphate metabolism, we have now identified D-myo-inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4), as the inositol phosphate most likely to mediate this uncoupling. A novel, membrane-permeant ester prepared by total synthesis delivers Ins(3,4,5,6)P4 intracellularly and confirms that this emerging messenger does inhibit Cl- flux resulting from thapsigargin- or histamine-induced [Ca2+]i elevations.

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)–induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3–only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector–mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal–regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim−/− mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.