Nuclear factor B (NF-B) and activator protein 1 (AP-1) are key transcription factors that orchestrate expression of many genes involved in inflammation, embryonic development, lymphoid differentiation, oncogenesis, and apoptosis (48, 62). NF-B and AP-1 activities are induced by a plethora of physiological and environmental stimuli (5, 51).The activity of NF-B is regulated by its interaction with the family of NF-B inhibitors known as IB, which results in the formation of inactive NF-B-IB complexes in the cytoplasm (3,4,60). In response to various stimuli, the IB kinase complex (IKK) then phosphorylates the IB bound to the NF-B complexes as substrates (8,36,45,47). The subsequent proteasome-mediated degradation of IB exposes the nuclear localization signal (NLS) of NF-B, which releases the NF-B proteins to be translocated to the nucleus, where they regulate the transcription of specific genes (5, 48).AP-1 is a group of basic leucine zipper (bZIP) transcription factors consisting of the Fos (c-Fos, FosB, Fra1, and Fra2) and Jun (c-Jun, JunB, and JunD) families (54, 62). The predominant forms of AP-1 in most cells are Fos/Jun heterodimers which have a high affinity for binding to an AP-1 site, whereas Jun/Jun homodimers bind to the AP-1 site with low affinity (54,62). A number of studies have shown that serum and growth factors that induce AP-1 do so by activating the extracellular signal-regulated kinase (ERK) subgroup of mitogen-activated protein kinases (MAPKs) (9, 27, 55). These activated members of MAPKs translocate to the nucleus to phosphorylate and thereby transcriptionally activate a subfamily of ETS domain transcription factors known as ternary complex factors (TCFs) that bind to fos promoters (9,27,55,65,80). fos, fosB, and other members of the AP-1 family of transcription factors are mainly regulated at their transcription through serum responsive elements (SREs) in their promoters (57,76). For example, the regulation of c-fos expression is controlled by Elk, a member of TCFs that associates with the serum response factor (SRF) (11,28,49). The elk-1 gene encodes two spliced variants: elk-1 and an alternatively spliced variant known as ⌬elk-1, which is missing the SRF interaction domain and part of the elk-1 DNA binding domain (61). The ⌬Elk-1protein cannot form an SRF-dependent ternary complex with SRE to activate fos transcription (61). However, a variety of experiments have shown that Elk-1 proteins play a central role in the response of cells to many extracellular signals and control the expression of genes involved in cell cycle progression, differentiation, and apoptosis (62, 75). The mechanism by which Elk-1 activates transcription in response to various stimuli has been extensively studied; however, less is known about the regulation of elk-1 gene expression itself.Even though NF-B and AP-1 transcription factors are regulated by different mechanisms, they appear to be activated simultaneously by the same multitude of stimuli (1,19,37,43,71,78). A number of reports also showed that these transcrip...

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the ␤1 and the ␣2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.

The transcription factor NF-kappaB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-kappaB in both transient and persistent phases. We investigated the mechanism for this biphasic NF-kappaB activation. Our results show that MEKK3 is essential in the regulation of rapid activation of NF-kappaB, whereas MEKK2 is important in controlling the delayed activation of NF-kappaB in response to stimulation with the cytokines TNF-alpha and IL-1alpha. MEKK3 is involved in the formation of the IkappaBalpha:NF-kappaB/IKK complex, whereas MEKK2 participates in assembling the IkappaBbeta:NF-kappaB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-kappaB activation. Thus, our study reveals a novel mechanism in which different MAP3K and IkappaB isoforms are involved in specific complex formation with IKK and NF-kappaB for regulating the biphasic NF-kappaB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression.

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The Rel/NF-kappaB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IkappaBalpha (IkappaBalphaM). In this report, we show that the inhibition of constitutive NF-kappaB activity, either by ectopic expression of IkappaBalphaM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IkappaBalpha proteins, downregulates the expression of bcl-xl. We identified two putative NF-kappaB binding sites (kappaB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-kappaB proteins. p65/p50 heterodimer interacts with kappaB/A site whereas p50/p50 homodimer interacts with kappaB/B. The bcl-xl promoter reporter gene assays reveal that NF-kappaB dependent transcriptional activation is mainly mediated by kappaB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IkappaBalphaM and PS-341 completely abolish NF-kappaB DNA binding activity; however, PS-341, but not ectopic expression of IkappaBalphaM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IkappaBbeta and the re-expression of NF-kappaB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-kappaB/IkappaB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-kappaB : IkappaBalpha and NF-kappaB : IkappaBbeta complexes are regulated by different upstream activators, and that NF-kappaB plays a key role in pancreatic tumorigenesis.

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright ؊/؊ embryos die at midgestation from failed hematopoiesis. Bright ؊/؊ embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright ؊/؊ mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.The formation and maintenance of blood throughout fetal and adult life rely on the self-renewal of hematopoietic stem cells (HSCs). Rare HSCs arise in the embryonic yolk sac and aorta-gonad mesonephros AGM, seed the fetal liver, and then circulate in the bone marrow of adult mammals. Fetal and adult HSC progenitors become progressively dedicated to differentiation into erythrocytes, myeloid cells, and lymphocytes. Transcription factors critical for the specification and formation of HSCs cover a wide range of DNA binding protein families. An emerging theme is that many of these same regulators are required later for the differentiation of individual blood lineages, which explains why a number of HSC transcription factors were discovered and originally characterized because of their deregulation in hematopoietic malignancies.Bright/Arid3a/Dril1 is the founder of the AT-rich interaction domain (ARID) superfamily of DNA binding proteins (18,60). Bright, in a complex with Bruton's tyrosine kinase (Btk) and TFII-I, binds to specific AT-rich motifs within the nuclearmatrix attachment regions (MARs) of the immunoglobulin heavy-chain (IgH) intronic enhancer (E) and selected IgH promoters to activate IgH transcription (18,25,30,43,44,55,57,58). B cell-specific, transgenic overexpression of Bright leads to partial blocks at both the late-pre-B and T1 immature stages, skewed marginal-zone (MZ) B cell development, increased natural IgM antibody production, and intrinsic autoimmunity (49). Transgenic dominant negative (DN) inhibition of Bright DNA binding results in reduced levels of IgM in serum and functional perturbation of IgM secretion by B-1 cells (39,48). A small pool of Bright cycles from the nucleus into plasma membrane lipid rafts, where it associates with Btk to dampen antigen receptor signaling (48).While highly B lineage restricted in...

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Both pro-and antiapoptotic activities of NF-B transcription factor have been observed; however, less is known about the mechanism by which NF-B induces apoptosis. To elucidate how NF-B regulates proapoptotic signaling, we performed functional analyses using wild-type, ikk1؊/؊ , ikk2 ؊/؊ , rela ؊/؊ murine fibroblasts, MDAPanc-28/Puro, MDAPanc-28/IB␣M, and HCT116/p53 ؉/؉ and HCT116/p53 ؊/؊ cells with investigational anticancer agent doxycycline as a superoxide inducer for generating apoptotic stimulus. In this report, we show that doxycycline increased superoxide generation and subsequently activated NF-B, which in turn up-regulated p53 expression and increased the stability and DNA binding activity of p53. Consequently, NF-Bdependent p53 activity induced the expression of p53-regulated genes PUMA and p21 waf1 as well as apoptosis. Importantly, lack of RelA, IKK, and p53 as well as expression of a dominant negative IB␣ (IB␣M) inhibited NF-B-dependent p53 activation and apoptosis. The doxycycline-induced NF-B activation was not inhibited in HCT116/p53 ؊/؊ cells. Our results demonstrate that NF-B plays an essential role in activation of wild-type p53 tumor suppressor to initiate proapoptotic signaling in response to overgeneration of superoxide. Thus, these findings reveal a mechanism of NF-B-regulated proapoptotic signaling.