An extended core nanocoax pillar architecture for enhanced molecular detection.

Biosensors that incorporate nanomaterials and nanofabrication techniques enable molecular detection of chemical and biological macromolecules with a high degree of specificity and ultrasensitivity. Here, we present a novel fabrication process that yields a nanostructure capable of detecting biological macromolecules. The extended core nanocoax (ECC) structure builds on a previously reported nanocoaxial-based sensor. The fabrication of the device incorporates an extended inner pillar, with controllable extension above the annulus and into the surrounding solution. This new design eliminates structural constraints inherent in the original nanocoax architecture. We also provide results demonstrating improvement in biosensing capability. Specifically, we show the capability of the new architecture to detect the B subunit of the Vibrio cholerae toxin at improved sensitivity (100 pg/ml) in comparison to optical enzyme-linked immunosorbant assay (1 ng/ml) and previously reported coaxial nanostructures (2 ng/ml).

Assembly of multi-functional nanocomponents on periodic nanotube array for biosensors.

Patterned carbon nanotubes arrays (PCNTA) with reduced density and length were developed with polystyrene sphere masked catalyst dots followed by plasma enhanced chemical vapor deposition method. The nanotubes were then uniformly coated with electropolymerized polypyrrole (PPy). The coating thickness was conformally adjustable. Gold nanoparticles (AuNP) together with glucose oxidase (Gox) were doped into the PPy film on the nanotubes to develop a high performance PCNTA glucose sensor. The sensitivity of the sensor was improved by the co-existence of Gox and AuNP on the carbon nanotube. Moreover, in contrast to previous reported PCNTA glucose sensors, the design herein utilized the entire surface of nanotubes as active sensing areas in order to maximize the Faradic currents. This research outlines a practical avenue to fabricate high performance PCNTA sensor chips with multiple molecules and functional nano-architectures.

Cutting edge: differential signaling requirements for activation of assembled cyclin D3-cdk4 complexes in B-1 and B-2 lymphocyte subsets.

B-1 lymphocytes represent a distinct B cell subset with unusual mitogenic responses. PMA alone promotes proliferation in B-1 cells, but not in splenic B-2 cells. Although cyclin D2-cyclin-dependent kinase 4 (cdk4) complexes mediate early retinoblastoma gene product (pRb) phosphorylation in B-1 cells, the transient nature of their accumulation cannot account for the continued increase in pRb phosphorylation, which is maximal at 24 h. We show herein that PMA promotes the accumulation of functional cyclin D3-cdk4 complexes in B-1 cells following loss of cyclin D2. PMA also induces accumulation of cyclin D3-cdk4 complexes in B-2 cells; however, these complexes do not phosphorylate pRb. Thus, PMA is sufficient to induce synthesis and assembly of cyclin D3-cdk4 complexes in B-1 and B-2 cells; however, PMA triggers cyclin D3-cdk4 activation only in B-1 cells. These results reveal a novel regulatory step that controls activation of cyclin D3-cdk4 complexes whose function segregates differentially in B cell subsets.

Rescue of CH31 B cells from antigen receptor-induced apoptosis by inhibition of p38 MAPK.

CH31 B lymphomas represent a model for antigen-induced deletional tolerance of immature B lymphocytes, because cross-linking the B cell antigen receptor (BCR) induces G(1) phase arrest and apoptosis. We have recently demonstrated that BCR cross-linking leads to a transient activation of p38 mitogen-activated protein kinase (MAPK) in CH31 B cells. In this paper, we functionally characterize the role of p38 MAPK in BCR-induced apoptosis as well as evaluate the regulation of additional MAPKs by the BCR. We demonstrate that JNK and ERK activities are not affected by BCR cross-linking, suggesting that these MAPKs are not directly involved in initiating the apoptotic cascade. By contrast, we show that pretreatment of CH31 B cells with the highly specific p38 MAPK inhibitor SB203580 ablated both BCR-induced p38 MAPK activity and apoptosis. Pretreatment of CH31 cells with an inactive SB203580 analog, SB202474, did not prevent apoptosis. These findings establish a key role for p38 MAPK in antigen receptor-mediated apoptosis of CH31 B cells.

Identification of a membrane Ig-induced p38 mitogen-activated protein kinase module that regulates cAMP response element binding protein phosphorylation and transcriptional activation in CH31 B cell lymphomas.

The cAMP response element (CRE) binding protein (CREB) is emerging as a key regulatory factor of gene transcription in B lymphocytes; however, the postreceptor pathways that regulate CREB activity and CRE-dependent gene transcription remain largely undefined. We investigated B cell Ag receptor (BCR)-mediated phosphorylation and activation of CREB in the surface IgM+ CH31 B cell lymphoma, which undergoes Ag-dependent cell death. The activity of p38 mitogen-activated protein kinase (MAPK) was increased in response to BCR ligation. Phosphorylation of CREB on serine 133, a modification that positively regulates its trans-activation, was concomitantly increased. Inhibition of p38 MAPK by pretreating CH31 B cells with the highly specific bicyclic imidazole inhibitor, SB203580, reduced BCR-induced CREB phosphorylation. BCR cross-linking also led to increased MAPK-activated protein kinase-2 activity, an enzyme that lies immediately downstream from p38 MAPK; MAPK-activated protein kinase-2 immune complexes phosphorylated a peptide substrate containing the CREB serine 133 phosphoacceptor motif. Given the role of CREB in regulating junB gene expression in mature B lymphocytes, we examined whether p38 MAPK activity was necessary for CRE-dependent junB transcription in CH31 B cells. BCR ligation led to increased junB mRNA levels, which were significantly reduced in CH31 B cells pretreated with SB203580. Activation of a CRE-dependent junB promoter/chloramphenicol acetyltransferase (CAT) reporter gene by the BCR was also blocked by SB203580. Similarly, inhibition of p38 MAPK in surface IgM+ WEHI-231 B cell lymphomas resulted in reduced BCR-induced junB mRNA expression and junB promoter activation. The results implicate a p38 MAPK pathway in BCR-mediated CREB phosphorylation and junB transcriptional activation in B cell lymphomas.

B cell antigen receptor-mediated activation of cyclin-dependent retinoblastoma protein kinases and inhibition by co-cross-linking with Fc gamma receptors.

Cross-linking the B cell Ag receptor (BCR) to surface Fc receptors for IgG (Fc gamma R) inhibits G1-to-S progression; the mechanism by which this occurs is not completely known. We investigated the regulation of three key cell cycle regulatory components by BCR-Fc gamma R co-cross-linking: G1-cyclins, cyclin-dependent kinases (Cdks), and the retinoblastoma gene product (Rb). Rb functions to suppress G1-to-S progression in mammalian cells. Rb undergoes cell-cycle-dependent phosphorylation, leading to its inactivation and thereby promoting S phase entry. We demonstrate in this paper for the first time that BCR-induced Rb phosphorylation is abrogated by co-cross-linking with Fc gamma R. The activation of Cdk4/6- and Cdk2-dependent Rb protein kinases is concomitantly blocked. Fc gamma R-mediated inhibition of Cdk2 activity results in part from an apparent failure to express Cdk2 protein. By contrast, inhibition of Cdk4/6 activities is not due to suppression of Cdk4/6 or cyclins D2/D3 expression or inhibition of Cdk-activating kinase activity. Cdk4- and Cdk6-immune complexes recovered from B cells following BCR-Fc gamma R co-cross-linking are devoid of coprecipitated D-type cyclins, indicating that inhibition of their Rb protein kinase activities is due in part to the absence of bound D-type cyclin. Thus, BCR-derived activation signals that up-regulate D-type cyclin and Cdk4/6 protein expression remain intact; however, Fc gamma R-mediated signals block cyclin D-Cdk4/6 assembly or stabilization. These results suggest that assembly or stabilization of D-type cyclin holoenzyme complexes 1) is an important step in the activation of Cdk4/6 by BCR signals, and 2) suffice in providing a mechanism to account for inhibition of BCR-stimulated Rb protein phosphorylation by Fc gamma R.

Early induction of cyclin D2 expression in phorbol ester-responsive B-1 lymphocytes.

B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2-4 h, in phorbol ester-stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester- stimulated B-2 cells. Phorbol ester-stimulated cyclin D2 expression was accompanied by the formation of cyclin D2-Cdk4, and, to a lesser extent, cyclin D2-Cdk6, complexes; cyclin D2- containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset.

Transient stimulation of the c-Jun-NH2-terminal kinase/activator protein 1 pathway and inhibition of extracellular signal-regulated kinase are early effects in paclitaxel-mediated apoptosis in human B lymphoblasts.

We demonstrate here that paclitaxel exposure to RPMI-1788 B lymphoblasts caused a dose- and time-dependent increase in nuclear factor activator protein 1 (AP-1) DNA binding activity. The basal DNA binding activities of nuclear factors NF-kappaB and Ets were not affected by paclitaxel. Consistent with these biochemical events, paclitaxel stimulated AP-1-dependent chloramphenicol acetyltransferase (CAT) reporter gene transcription in vivo, as directed from a tetradecanoyl phorbol acetate-inducible promoter. AP-1 binding activity of nuclear extracts isolated from paclitaxel treated cells was reduced following immunodepletion with antibodies directed against individual Jun family proteins, whereas anti-cFos, anti-Fra1, and anti-FosB antibodies were not inhibitory. Paclitaxel caused a rapid and transient increase in c-Jun NH2-terminal kinase (JNK) activity, a proposed mediator of stress activation pathways. By contrast, exposure to paclitaxel produced a transient reduction in the extracellular signal-regulated mitogen-activated protein kinase 2 (ERK2) activity, a proposed mediator of growth factor-stimulated proliferation pathways. Transient activation of the c-Jun-NH2-terminal kinase/AP-1 pathway, together with down-regulation of ERK2 activity, may be a key event in the early response of RPMI-1788 B lymphoblasts to paclitaxel exposure.

Transcriptional regulation of the junB gene in B lymphocytes: role of protein kinase A and a membrane Ig-regulated protein phosphatase.

We have examined herein whether membrane Ig (mIg) stimulates junB transcription through a protein kinase A (PKA)-dependent or PKA-independent pathway. PKA phosphotransferase activity was not increased following mIg cross-linking of Bal17 B cells. However, junB transcriptional activation was dependent upon PKA activity, as evidenced by inhibition of goat anti-mouse IgM-stimulated junB promoter-chloramphenicol acetyltransferase reporter gene activity in transfected Bal17 B cells treated with the PKA inhibitor H-89. mIg-stimulated junB promoter-chloramphenicol acetyltransferase activity was also blocked in B cells expressing a specific PKA inhibitor peptide, whereas in vivo expression of an inactive PKA inhibitor peptide variant was not inhibitory. Expression of a mutant cAMP response element binding protein (CREB) containing an inactivated kinase A phosphoacceptor site at Ser133 reduced mIg-stimulated junB transcription. Okadaic acid increased CREB1 phosphorylation at Ser133 and junB transcriptional activation, suggesting the action of protein phosphatase-1 (PP-1) or -2A (PP-2A). Extracts from unstimulated B cells exhibited phosphatase activity against an in vitro PKA-phosphorylated peptide containing the Ser133 phosphoacceptor site. The involvement of a phosphatase activity in regulating mIg-stimulated junB transcription is supported by our finding that extracts from goat anti-mouse IgM-stimulated B cells exhibited a significantly reduced level of Ser133 phosphatase activity. Hence, the level of CREB1 phosphorylation is governed by the balance between PKA and phosphatase activities. junB transcriptional activation results in part from mIg signals that negatively regulate a CREB1-targeted PP-1 or PP-2A activity.

Transcriptional regulation of the junB promoter in mature B lymphocytes. Activation through a cyclic adenosine 3',5'-monophosphate-like binding site.

The experiments presented herein were designed to understand the molecular mechanism(s) by which membrane Ig (mIg)-dependent signals are integrated at the level of the junB promoter to induce gene transcription. Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequences identified a region located between -194 and -87 that contains an Ets binding site and a putative cAMP response element binding site (CRE-like). Point mutagenesis of the CRE-like site blocked junB promoter activation in response to mIg cross-linking in mature Bal17 B cells. Nuclear extract binding activity to a synthetic oligonucleotide containing the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was competed with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif, the latter observation suggests that members of the activating transcription factor/CRE binding protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating transcription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negative CREB protein blocked mIg-mediated transcription from a junB CRE-like site-chloramphenicol acetyltransferase reporter gene. CRE-like nucleoprotein complexes from Bal17 B cells contained constitutively bound CREB-1, which was phosphorylated on serine 133 in response to mIg cross-linking. Activating transcription factor-1 protein was also constitutively expressed in CRE-like nucleoprotein complexes. Collectively, these results suggest that components of the protein kinase A signaling pathway are recruited by mIg to induce junB transcription.

Regulation of the catalytic subunit (p34PSK-J3/cdk4) for the major D-type cyclin in mature B lymphocytes.

We examined the expression of the cyclin-dependent kinase 4, p34PSK-J3/cdk4 protein, in small dense, activated, and proliferating primary B lymphocytes. A small steady state level of cdk4 synthesis was detected in resting B cells. Stimulation of resting B cells with mitogenic amounts of F(ab')2 fragments of goat anti-mouse IgM (anti-Ig) resulted in increased synthesis of cdk4 protein during the mid to late G1 phase of the cell cycle; LPS or the combination of phorbol ester and calcium ionophore also elevated cdk4 levels. Resting B cells that we rendered competent by treatment with IL 4 or low doses of anti-Ig or, alternatively, were activated by phorbol ester or ionomycin alone also exhibited heightened cdk4 protein levels. Subsequent analysis of potential cdk4 regulatory subunit D-type cyclins revealed that cyclin D2, not cyclin D1 or D3, is expressed in primary mature B lymphocytes. The induction of cyclin D2 synthesis in response to mitogenic anti-Ig paralleled cdk4 expression; however, IL-4 or low dose anti-Ig alone did not increase the rate of de novo cyclin D2 synthesis above that of resting B cells. The significance of the lack of cyclin D2 regulation by competence-inducing growth factors was demonstrated, in that only mitogenic factors that stimulated DNA synthesis 1) led to the formation of stable cyclin D2/cdk4 holoenzyme complexes during G1 phase progression, and 2) afforded the isolation of anti-cyclin D2 or anti-cdk4 immunoprecipitates that phosphorylated retinoblastoma. These findings suggest a role for these proteins during the mid to late G1 phase progression and possibly the G1/S phase transition in primary mature B lymphocytes.

Membrane immunoglobulin receptor cross-linking induces fosB mRNA expression in mature B lymphocytes: assembly of distinct FosB-containing nucleoprotein complexes during B cell stimulation.

We demonstrate herein that anti-Ig stimulation of Bal-17 B cells leads to a rapid and transient increase in fosB mRNA levels, with kinetics similar to those previously reported for members of the jun gene family. The coupling of fosB expression to known protein kinases that are activated following membrane immunoglobulin receptor (mIg) cross-linking was evaluated. Inhibition of src-protein tyrosine kinase activity by pretreatment of Bal-17 B cells with herbimycin A prevented subsequent anti-Ig-dependent increases in fosB mRNA levels. Moreover, inhibition of protein kinase C (PKC) by pretreatment of Bal-17 B cells with staurosporine, H7, or by phorbol ester-induced down-regulation of PKC activity blocked anti-Ig-stimulated fosB gene expression. These findings suggest that fosB expression is coupled to a mIg-mediated pathway that utilizes activated src-protein tyrosine kinases and PKC. Immunoblotting of Bal-17 B cell extracts with anti-FosB antiserum revealed that mIg cross-linking results in an increase in FosB protein levels. Moreover, immunoprecipitation of nondenatured Bal-17 B cell extracts indicated that FosB forms complexes in vivo with JunB and JunD and to a lesser extent with cJun. The anti-Ig-induced FosB/Jun complexes bind to several distinct cis-acting DNA elements, including AP-1 and NF-AB, which have been implicated in regulating nuclear gene expression during B cell activation. Collectively, these results suggest that FosB plays a central role in regulating gene expression during anti-Ig-mediated B cell activation.

Activation of AP-1 in primary B lymphocytes by surface immunoglobulin requires de novo Jun-B synthesis.

We demonstrate herein that resting primary B lymphocytes do not contain detectable levels of AP-1 (TRE)-binding activity. Upon cross-linking of surface immunoglobulin (sIg) receptors, TRE-binding activity is induced within 2 hr and its appearance requires de novo protein synthesis. Antisera to Jun-B inhibits the vast majority of TRE-binding activity, indicating that Jun-B is a primary component of B cell TRE-binding complexes. In keeping with this, Jun-B protein is not detectable in cytosol or nuclear extracts from resting B lymphocytes, as determined by immunoblotting with Jun-B antisera. However, the nuclear expression of Jun-B is induced within 2 hr following sIg cross-linking and is completely blocked by cycloheximide. 35S-labeling studies suggest that the increase in Jun-B expression results from de novo protein synthesis. Moreover, Jun-B migrates in SDS-polyacrylamide gels as two distinct electrophoretic proteins that correspond to a 41-kDa species and a phosphorylated 47-kDa form. These results suggest that the induction of AP-1-binding activity in primary B lymphocytes following sIg cross-linking does not result from post-translational phosphorylation of a preexisting cellular pool of Jun-B protein, but rather is coupled to the stimulation of de novo Jun-B synthesis. Thus Jun-B synthesis represents an integral event in the production of receptor-mediated AP-1 in B cells. The significance of these results with respect to the role of Jun-B in controlling gene expression during the activation of primary B cells is discussed.

Cell cycle-specific induction of Cdk2 expression in B lymphocytes following antigen receptor cross-linking.

The ligation of membrane Ig (mIg) on quiescent primary B lymphocytes by mitogenic concentrations of anti-IgM antibodies leads to cell cycle progression. The level of cyclin-dependent kinase 2 (Cdk2) expression was found to be restricted to specific phases of the cell cycle in primary cultures of murine B lymphocytes. Resting G0 phase, G1 phase, or B cells arrested near the G1/S boundary by hydroxyurea contained no detectable Cdk2 protein or associated histone H1 kinase activity. In contrast, B cell entry into S phase was accompanied by an induction in the expression of cellular Cdk2 as judged by immunoblotting of B cell lysates with anti-Cdk2 antibodies. Concomitant with S phase entry was the detection of anti-Cdk2-specific immunoprecipitable histone H1 kinase activity. Further analysis revealed that the amount of cyclin A protein also oscillated during cell cycle, appearing initially in G1 phase B cells. Cyclin A was found to be associated with Cdk2 in B cells during S phase progression. These results indicate that cross-linking of mIg on primary B lymphocytes results in the "downstream" catalytic activation of Cdk2. The timing of Cdk2 expression and its association with cyclin A suggests that Cdk2 may not be involved in the decision to enter S phase, but rather may provide a role in the maintenance of S phase progression or in preparing B cells to enter M phase.

Induction of CREB activity via the surface Ig receptor of B cells.

The regulation and function of CREB was examined in B cells to begin to elucidate the role of cAMP-derived signals in B cell activation. CRE-binding activity detected by the electrophoretic mobility shift assay was found to be constitutively expressed in nuclear extracts of primary murine splenic B cells and was unchanged in nuclear extracts obtained from B cells stimulated in a variety of ways. This activity was shown to be specific by competition analysis and to represent CREB or a closely related molecule on the basis of a "supershift" in the mobility of the nucleoprotein complex induced by anti-CREB antiserum. The function of B cell CREB was assessed by transient transfection of the murine B lymphoma cell line, BAL-17, with a CRE-dependent chloramphenicol acetyl-transferase (CAT) construct that contains a portion of the somatostatin promoter. Cross-linking of the surface Ig receptors of transfected BAL-17 B cells produced a threefold induction of CAT activity. Forskolin, which markedly induced CAT expression in PC12 cells transfected with the CRE-dependent construct, failed to stimulate CAT activity in transfected BAL-17 B cells despite an increase in cAMP. However, anti-Ig was found to act in synergy with forskolin to produce enhanced CAT activity. A phosphoprotein of appropriate molecular size for CREB was immunoprecipitated from anti-Ig plus forskolin treated BAL-17 B cells. These results suggest that CREB is present in primary B cells and that CRE-dependent gene expression is regulated by surface Ig either alone or in synergy with cAMP; the latter implies cross-talk between intracellular signaling pathways acting at the level of CREB.

Enhanced mRNA content in response to amino acid starvation for a 73 kDa protein of the inner mitochondrial membrane.

Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepatocytes or hepatoma cells. One of these proteins, MP-73, was synthesized at a rate 2- to 3-fold greater when cells were incubated for 3-9 h under conditions of amino acid deprivation versus amino acid supplementation. Immunoblotting with polyclonal antibodies prepared against MP-73 localized it to the inner mitochondrial membrane. MP-73 appears to be a hydrophobic, integral membrane protein. MP-73 antibody was used to identify a partial cDNA (NS3.2) of approximately 2 kb. A probe prepared from pNS3.2 identified a transcript in rat Fao hepatoma cells of approximately 4.4 kb that was increased in abundance by more than 20-fold following amino acid starvation of the cells.

T cell-dependent induction of NF-kappa B in B cells.

In comparison to B cell stimulation mediated by surface immunoglobulin (Ig) antigen receptor ligation, little is known about the intracellular events associated with T cell-dependent B cell responses. A model for the efferent phase of T cell-B cell interaction was used to examine the capacity of activated T cells to trigger nuclear expression of the trans-acting transcription factor, NF-kappa B, in B cells. Fixed, activated, but not fixed, resting Th2 cells were found to induce increased binding activity for a kappa B site-containing oligonucleotide in a time-dependent manner. This induction of NF-kappa B was eliminated by an antibody directed against a 39-kD cell interaction protein on activated T cells as well as by a soluble form of B cell CD40. Of particular relevance to intracellular signaling, NF-kappa B induction was not diminished by prior depletion of B cell protein kinase C (PKC) with phorbol myristate acetate. These results strongly suggest that T cell-dependent B cell stimulation is associated with NF-kappa B induction via p39-CD40 interaction and that this is brought about by non-PKC dependent signaling, in marked contrast to the previously documented requirement for PKC in sIg receptor-mediated stimulation. This suggest that NF-kappa B responds to more than one receptor-mediated intracellular signaling pathway in B cells and may be part of a "final common pathway" for B cell stimulation.

Surface Ig receptor-induced nuclear AP-1-dependent gene expression in B lymphocytes.

The relationship between signals generated via the sIgR complex of B lymphocytes and subsequent changes in gene expression is poorly understood at the molecular level. To illuminate mechanisms that may couple these events, we examined the expression and function of tetradecanoyl phorbol acetate-response element (TRE)-binding proteins (i.e., activator protein 1, (AP-1)) in the murine B lymphoma cell line BAL-17.7.1 (BAL-17), which models primary B lymphocyte responses in a number of respects. Cross-linking of sIgR led to substantial induction of nuclear AP-1, in BAL-17 B cells, that bound the TRE, as detected by electrophoretic mobility shift assay. The sIgR-induced TRE-binding activity consisted of both Jun and Fos proteins, on the basis of immunoreactivity of nucleoprotein complexes with specific antisera. In addition, immunoprecipitation with specific antisera showed that de novo synthesis of Jun-B and c-Jun proteins, accompanied by c-Fos, was stimulated after cross-linking of sIgR on BAL-17 B cells. Transient transfection of BAL-17 B cells with reporter gene constructs showed that B cell AP-1 failed to trans-activate the TRE-containing human collagenase gene promoter, for which activity is dependent upon functional expression of cellular c-Jun. In contrast, sIg-induced AP-1 trans-activated a HSV-tk promoter that contained three TRE; this pattern of gene expression is consistent with the presence of functional Jun-B-containing AP-1 in B lymphocytes. These results are the first to attribute a functional role to sIgR-mediated AP-1 in B lymphoid cells and suggest that AP-1 functions to couple the sIgR complex to changes in nuclear gene expression.

Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface Ig receptor.

Constitutive expression of NF-kappa B has been associated with developmental maturity in B cells on the basis of studies using continuously growing cell lines and plasmacytomas; however, little is known about the behavior of NF-kappa B in primary, mature B cells. In the present work, the regulation of NF-kappa B expression was studied by analyzing subcellular fractions of adult murine splenic B cells with the electrophoretic mobility shift assay using a kappa B-containing oligonucleotide. Although nuclear extracts from resting B cells contained kappa B-binding activity, additional kappa B-binding activity was present in cytosolic fractions in a form that became apparent after treatment with detergent. Competition analysis indicated that the DNA binding activity detected by electrophoretic gel mobility shift assay was specific for the kappa B motif, and UV photo-cross-linking showed the molecular size of kappa B-binding protein to be similar to that of the DNA binding subunit of NF-kappa B. Nuclear expression of kappa B-binding activity was markedly induced by treatment of B cells with phorbol ester or with LPS. Most notably, kappa B-binding activity was induced after surface IgR cross-linking, and the mechanism of this induction involved PKC. Further, anti-Ig-induced activity was superinduced in the presence of cycloheximide. These results indicate that nuclear NF-kappa B is rapidly induced as a result of B cell stimulation, and further suggest that NF-kappa B may play a specific role in mature B cells after ligand binding to surface Ig distinct from its postulated developmental role as a stage-specific factor involved in kappa-enhancer function.