Sex reversal by loss of the C-terminal transactivation domain of human SOX9.

Haploinsufficiency for SOX9 has recently been identified as the cause for both campomelic dysplasia (CD), a human skeletal malformation syndrome, and the associated autosomal XY sex reversal. SOX9 contains a putative DNA-binding motif known as the high-mobility group (HMG) domain characterizing a whole class of transcription factors. We show in cell transfection experiments that SOX9 can transactivate transcription from a reporter plasmid through the motif AACAAAG, a sequence recognized by other HMG domain transcription factors. By fusing all or part of SOX9 to the DNA-binding domain of yeast GAL4, the transactivating function was mapped to a transcription activation (TA) domain at the C terminus of SOX9. This non-acidic TA domain is evolutionarily conserved and rich in proline, glutamine and serine. With one exception, all SOX9 nonsense and frame shift mutations described so far in CD/sex reversal patients lead to truncation of the TA domain, suggesting that impairment of gonadal and skeletal development in these cases results, at least in part, from loss of transactivation of genes downstream of SOX9.

Phase IB study of the EpCAM antibody adecatumumab combined with docetaxel in patients with EpCAM-positive relapsed or refractory advanced-stage breast cancer.

BACKGROUND: Targeted therapy options in HER2-negative breast cancer are limited. This open-label, multicenter phase IB dose-escalation trial was conducted to determine safety, tolerability, and antitumor activity of a combination of docetaxel (Taxotere) and increasing doses of adecatumumab, a human IgG1 antibody targeting epithelial cell adhesion molecule (EpCAM), in EpCAM-positive relapsed or primary refractory advanced-stage breast cancer. PATIENTS AND METHODS: Patients pretreated with up to four prior chemotherapy regimens received increasing adecatumumab doses either every 3 weeks (q3w) or weekly (qw) combined with docetaxel (100 mg/m(2) q3w). Primary end points were safety and tolerability. Antitumor activity was evaluated according to RECIST. Clinical benefit was defined as complete or partial response or stable disease for ≥24 weeks. RESULTS: Thirty-one evaluable patients were treated. Most adverse events were mild to moderate in severity. Neutropenia, leukocytopenia, lymphopenia, and diarrhea (dose-limiting) were the most frequent toxic effects. Maximum tolerated doses of adecatumumab given in combination with docetaxel were 550 mg/m(2) q3w and 360 mg/m(2) qw. Clinical benefit was observed in 44% of patients treated with q3w adecatumumab and docetaxel, increasing to 63% in patients with high EpCAM-expressing tumors. CONCLUSION: Combination therapy of adecatumumab and docetaxel is safe, feasible, and potentially active in heavily pretreated advanced-stage breast cancer.

Control of gene expression by proteolysis.

The proteasome and the small protein ubiquitin are key elements in the intracellular pathway of general protein degradation. Recent evidence shows that the proteasome and other less well defined cytoplasmic proteases can participate in specific events which control inducible gene expression. A number of eukaryotic transcriptional regulators, including NF-kappa B/l kappa B, p53, c-Jun, Notch, sterol regulated element binding proteins and MAT2 alpha, have recently been shown to be regulated by proteolytic events, a regulation which results in the activation or inactivation of gene expression.

KBF1 (p50 NF-kappa B homodimer) acts as a repressor of H-2Kb gene expression in metastatic tumor cells.

Downregulation of major histocompatibility complex class I expression is causally related to high malignancy and low immunogenicity of certain murine tumors. In this study, we have analyzed the roles of the nuclear factors KBF1/p50 and p65 in regulation of class I expression in high and low metastatic tumor cells. Low class I-expressing cells show at higher levels of KBF1/p50 and NF-kappa B (p50/p65) binding activity than high class I-expressing cells. However, an excess of KBF1 over NF-kappa B is observed in low expressing cells, while an excess of NF-kappa B over KBF1 is observed in high expressing cells. Stable transfection of a p65 expression vector into low class I-expressing cells activated H-2 transcription and cell surface expression, while stable transfection of p50 expression vector into high expressing cells suppressed H-2Kb transcription and cell surface expression. Our studies suggest that KBF1 has the potential of downregulating class I gene expression, whereas dimers containing the p65 subunit are activators of class I gene expression.

Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells.

Dithiocarbamates and iron chelators were recently considered for the treatment of AIDS and neurodegenerative diseases. In this study, we show that dithiocarbamates and metal chelators can potently block the activation of nuclear factor kappa B (NF-kappa B), a transcription factor involved in human immunodeficiency virus type 1 (HIV-1) expression, signaling, and immediate early gene activation during inflammatory processes. Using cell cultures, the pyrrolidine derivative of dithiocarbamate (PDTC) was investigated in detail. Micromolar amounts of PDTC reversibly suppressed the release of the inhibitory subunit I kappa B from the latent cytoplasmic form of NF-kappa B in cells treated with phorbol ester, interleukin 1, and tumor necrosis factor alpha. Other DNA binding activities and the induction of AP-1 by phorbol ester were not affected. The antioxidant PDTC also blocked the activation of NF-kappa B by bacterial lipopolysaccharide (LPS), suggesting a role of oxygen radicals in the intracellular signaling of LPS. This idea was supported by demonstrating that treatment of pre-B and B cells with LPS induced the production of O2- and H2O2. PDTC prevented specifically the kappa B-dependent transactivation of reporter genes under the control of the HIV-1 long terminal repeat and simian virus 40 enhancer. The results from this study lend further support to the idea that oxygen radicals play an important role in the activation of NF-kappa B and HIV-1.

T cell leukemia-associated human Notch/translocation-associated Notch homologue has I kappa B-like activity and physically interacts with nuclear factor-kappa B proteins in T cells.

Translocation-associated Notch homologue (TAN-1), a gene originally cloned from the translocation breakpoint of a human T cell leukemia carrying a 9:7(q34.3) translocation, encodes a protein belonging to the Notch/Lin-12/Glp-1 receptor family. These receptors mediate the specification of numerous cell fates during development in invertebrates and vertebrates. The intracellular portion of Notch/TAN-1 contains six ankyrin repeats that are similar to those found in cytoplasmic I kappa B proteins. I kappa B proteins are specific inhibitors of nuclear factor (NF)-kappa B/Rel transcription factors. Here we show that TAN-1 has functional properties of an I kappa B-like regulator with specificity for the NF-kappa B p50 subunit. A recombinant polypeptide corresponding to the cytoplasmic portion of TAN-1 (TAN-1C) specifically inhibited the DNA binding of p50-containing NF-kappa B complexes. When overexpressed in an appropriate cell line, TAN-1C prevented kappa B-dependent transactivation in transient reporter gene assays in a fashion similar to the structurally related protein, Bcl-3. TAN-1C could activate kappa B-dependent gene expression by attenuating the inhibitory effect of an excess of p50 homodimers. Immunoprecipitation experiments showed that the TAN-1 from a T cell line is associated with NF-kappa B containing p50 and p65 subunits. These observations indicate that TAN-1C may directly engage NF-kappa B transcription factors and modulate nuclear gene expression.

The immunosuppressive fungal metabolite gliotoxin specifically inhibits transcription factor NF-kappaB.

Opportunistic infections, such as aspergillosis, are among the most serious complications suffered by immunocompromised patients. Aspergillus fumigatus and other pathogenic fungi synthesize a toxic epipolythiodioxopiperazine metabolite called gliotoxin. Gliotoxin exhibits profound immunosuppressive activity in vivo. It induces apoptosis in thymocytes, splenocytes, and mesenteric lymph node cells and can selectively deplete bone marrow of mature lymphocytes. The molecular mechanism by which gliotoxin exerts these effects remains unknown. Here, we report that nanomolar concentrations of gliotoxin inhibited the activation of transcription factor NF-kappaB in response to a variety of stimuli in T and B cells. The effect of gliotoxin was specific because, at the same concentrations, the toxin did not affect activation of the transcription factor NF-AT or of interferon-responsive signal transducers and activators of transcription. Likewise, the activity of the constitutively DNA-binding transcription factors Oct-1 and cyclic AMP response element binding protein (CREB), as well as the activation of protein tyrosine kinases p56lck and p59fyn, was not altered by gliotoxin. Very high concentrations of gliotoxin prevented NF-kappaB DNA binding in vitro. However, in intact cells, inhibition of NF-kappaB did not occur at the level of DNA binding; rather, the toxin appeared to prevent degradation of IkappaB-alpha, NF-kappaB's inhibitory subunit. Our data raise the possibility that the immunosuppression observed during aspergillosis results in part from gliotoxin-mediated NF-kappaB inhibition.

Combined blockade of granulocyte-macrophage colony stimulating factor and interleukin 17 pathways potently suppresses chronic destructive arthritis in a tumour necrosis factor alpha-independent mouse model.

OBJECTIVE: A pathogenic role for granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)17 in rheumatoid arthritis (RA) has been suggested. In previously published work, the therapeutic potentials of GM-CSF and IL17 blockade in arthritis have been described. In the present study, the simultaneous blockade of both pathways in a mouse model for chronic arthritis was investigated to identify whether this double blockade provides a superior therapeutic efficacy. METHODS: A chronic relapsing arthritis was induced in C57Bl/6 wild type (WT) and C57Bl/6 genetically deficient for IL17 receptor (IL17R knockout (KO)) mice by intra-articular injection of Streptococcal cell wall (SCW) fragments into knees on days 0, 7, 14 and 21. Treatments (intraperitoneal) were given weekly starting on day 14. Animals were analysed for inflammation, joint damage and a range of inflammatory mediators. RESULTS: Joint swelling and cartilage damage were significantly reduced in the IL17R KO mice and in WT mice receiving anti-GM-CSF neutralising mAb 22E9 compared to isotype control antibodies. The therapeutic effect was significantly more pronounced in mice where IL17 and GM-CSF pathways were inhibited (eg, IL17R KO mice treated with 22E9 mAb). Tumour necrosis factor (TNF)alpha blockade had essentially no effect. CONCLUSION: Our data further support the therapeutic potentials of GM-CSF and IL17 blockade in a RA model that is no longer responsive to an established TNFalpha antagonist, moreover, our results suggest that concomitant inhibition of both pathways may provide the basis for a highly effective treatment of chronic RA in patients that are resistant to treatment by TNFalpha inhibitors.

A role for oxygen radicals as second messengers.

All cells seem to produce oxygen radicals. Recent results suggest that small nontoxic amounts of these radicals are released by various cell types in response to stimulation with tumour necrosis factor (TNF), interleukin 1 (IL-1) and phorbol esters, all of which activate a cytoplasmic form of the transcription factor NF-kappa B by releasing an inhibitory protein subunit. Activation is inhibited by agents that remove oxygen radicals, and mimicked by exposure to mild oxidant stress. This article proposes that oxygen radicals act as second messengers for a variety of agents, including the immunomodulatory cytokines TNF and IL-1, in at least one type of regulatory pathway activating NF-kappa B.

Proteins controlling the nuclear uptake of NF-kappa B, Rel and dorsal.

The two DNA-binding subunits of the transcription factor NF-kappa B, the products of the rel oncogene family and the product of the developmental control gene dorsal of Drosophila are homologous within a 300 amino acid region. This sequence represents a novel DNA-binding and dimerization domain. The access of the NF-kappa B/Rel/dorsal (NRD) transcription factor family to the cell nucleus is regulated. There is now evidence that functionally and structurally related accessory proteins of NF-kappa B, Rel and dorsal control the nuclear entry as well as DNA-binding activity of the transcription factors. This review summarizes current knowledge about the nuclear-uptake regulatory proteins (NURPs) I kappa B-alpha, I kappa B-beta/pp40 and cactus.

Endoplasmicreticulum-induced signal transduction and gene expression.

Cells can respond to perturbations in endoplasmic reticulum (ER) function by activating two distinct signal-transduction pathways: one responds to unfolded proteins, the other to an overload of the organelle with membrane proteins. A third pathway is activated upon sterol depletion of cells and involves the cleavage and subsequent nuclear translocation of an ER membrane-bound transcription factor. Thus, three distinct pathways each activated by a different signal are currently known to project from the ER into the nucleus. This review summarizes the current understanding of these three pathways.

Tyrosine sulfation is a trans-Golgi-specific protein modification.

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.

Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention.

We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF-kappaB. However, activation of both NF-kappaB DNA binding and kappaB-dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.

Bcl-2 down-regulates the activity of transcription factor NF-kappaB induced upon apoptosis.

Among the many target genes of the transcription factor NF-kappaB are p53 and c-myc, both of which are involved in apoptosis. This prompted us to investigate the role of NF-kappaB in this process. We report that NF-kappaB is potently activated upon serum starvation, a condition leading to apoptosis in 293 cells. Similar to Bcl-2, a transdominant-negative mutant of the NF-kappaB p65 subunit partially inhibited apoptosis, indicating a direct involvement of the transcription factor in induction of cell death. As expected, the p65 mutant suppresses kappaB-dependent gene expression. Surprisingly, transiently or stably overexpressed Bcl-2 had the same effect. The transcription inhibitory activity of the two proteins correlated with their cell death protective potential. Like Bcl-2, the related protein Bcl-xL but not Bcl-xS was able to suppress kB-dependent transcription. Bcl-2 inhibited NF-kappaB activity by an unusual mechanism. It did not prevent the release of IkappaB in the cytoplasm but down-modulated the transactivating potential of nuclear p65. These data show that NF-kappaB can participate in apoptosis. We suggest that at least part of the anti-apoptotic potential of Bcl-2 may be explained from a hitherto undiscovered activity of Bcl-2 in controlling nuclear gene expression.

I kappa B: a specific inhibitor of the NF-kappa B transcription factor.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein (I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated cells--in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-kappa B and I kappa B was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase C, cause dissociation of a cytoplasmic complex of NF-kappa B and I kappa B by modifying I kappa B. this releases active NF-kappa B which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.

Selective activation of NF-kappa B by nerve growth factor through the neurotrophin receptor p75.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activated the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation was not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3.

Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.

The sequence reported in our 22 March 1991 report "Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NFkappaB" [Science 251, 1490 (1991)], contained some errors. Resequencing under strong denaturing conditions revealed three insertions at nucleotide positions 1194, 1212, and 1220, which changed the AA sequence from RSAR-PRLGP to QISQASALAP (residues 372 to 380), thus accounting for some of the divergence in this region. A corrected sequence has been sent to GenBank.

Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B.

A DNA probe that spanned a domain conserved among the proto-oncogene c-rel, the Drosophila morphogen dorsal, and the p50 DNA binding subunit of NF-kappa B was generated from Jurkat T cell complementary DNA with the polymerase chain reaction (PCR) and degenerate oligonucleotides. This probe was used to identify a rel-related complementary DNA that hybridized to a 2.6-kilobase messenger RNA present in human T and B lymphocytes. In vitro transcription and translation of the complementary DNA resulted in the synthesis of a protein with an apparent molecular size of 65 kilodaltons (kD). The translated protein showed weak DNA binding with a specificity for the kappa B binding motif. This protein-DNA complex comigrated with the complex obtained with the purified human p65 NF-kappa B subunit and binding was inhibited by I kappa B-alpha and -beta proteins. In addition, the 65-kD protein associated with the p50 subunit of NF-kappa B and the kappa B probe to form a complex with the same electrophoretic mobility as the NF-kappa B-DNA complex. Therefore the rel-related 65-kD protein may represent the p65 subunit of the active NF-kappa B transcription factor complex.

The ER-overload response: activation of NF-kappa B.

Various conditions that perturb the function of the endoplasmic reticulum (ER) were recently shown to activate the transcription factor NF-kappa B. Activation of NF-kappa B is caused by the accumulation of proteins in the ER membrane, a condition we have called ER overload. Both the release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates are required for ER-overload-mediated NF-kappa B activation. This novel intracellular signal transduction pathway might be important in antiviral defense and play a role in various diseases as well as in B-cell development.

Pro-inflammatory signaling: last pieces in the NF-kappaB puzzle?

The discovery of three previously unknown kinases that are essential for signaling in response to tumour necrosis factor and interleukin-1 has provided the missing link in the pathway that activates inflammatory genes.