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Biomedical subjects

Sankar Ghosh

Publications and source records attributed to Sankar Ghosh.

At least 37 records · Page 2Linked to original sources
PubMed2005

NFkappaB activates in vivo the synthesis of inducible Cox-2 in the brain.

Interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression in many of its cellular targets resulting in production and release of prostaglandins. Although IL-1beta-induced Cox-2 expression most likely requires activation of nuclear transcription factor kappa B (NFkappaB) pathway, this has never been formally demonstrated in vivo. We tested this using a specific inhibitor of NFkappaB activation, the NEMO binding domain (NBD) peptide, that has been shown previously to be effective in various in vivo models of acute inflammation. Incubation of rat glioma cells with the NBD peptide blocked IL-1beta-induced NFkappaB nuclear translocation. Furthermore, after injection of a biotinylated version of the NBD peptide into the lateral ventricle of the brain, we found that it readily diffused to its potential cellular targets in vivo. To test the effects of the peptide on NFkappaB activation and Cox-2 expression in the brain, we injected it intracerebroventricularly (36 microg/rat) into rats before intraperitoneal injection of IL-1beta (60 microg/kg). Treatment with NBD peptide completely abolished IL-1beta-induced NFkappaB activation and Cox-2 synthesis in microvasculature. In contrast, the peptide had no effect on constitutive neuronal Cox-2. These findings strongly support the hypothesis that IL-1beta-induced NFkappaB activation plays a major role in transmission of immune signals from the periphery to the brain.

Animals↗
PubMed2005

Role of nuclear factor-kappaB in the immune system and bone.

Bone metabolism is regulated by hormonal or local factors in the bone microenvironment, and recent studies have revealed that bone homeostasis is also influenced by immune system. The term 'osteoimmunology' has been proposed to explain the cross-talk between bone and the immune system. A critical element in this cross-talk is the inducible transcription factor nuclear factor-kappaB (NF-kappaB), which regulates gene expression during inflammatory and immune responses. However, NF-kappaB-signaling pathways are also important for bone homeostasis, in particular for osteoclast differentiation. By bridging inflammation and bone homeostasis, NF-kappaB also contributes to the onset and progression of arthritis. Several natural compounds, synthetic drugs, and gene-transfer technologies that lead to inhibition of the inhibitor of NF-kappaB kinase (IKK)/NF-kappaB activation pathway can prevent arthritis in animal models. In this review, we discuss the signaling pathway that leads to NF-kappaB activation and the role of NF-kappaB on osteoclast differentiation. Furthermore, we discuss the possibility that inhibition of NF-kappaB might provide novel therapeutic approach for inhibiting bone destruction in rheumatoid arthritis.

Animals↗
PubMed2004

Signaling to NF-kappaB.

The transcription factor NF-kappaB has been the focus of intense investigation for nearly two decades. Over this period, considerable progress has been made in determining the function and regulation of NF-kappaB, although there are nuances in this important signaling pathway that still remain to be understood. The challenge now is to reconcile the regulatory complexity in this pathway with the complexity of responses in which NF-kappaB family members play important roles. In this review, we provide an overview of established NF-kappaB signaling pathways with focus on the current state of research into the mechanisms that regulate IKK activation and NF-kappaB transcriptional activity.

Animals↗
PubMed2004

A novel ubiquitin-like domain in IkappaB kinase beta is required for functional activity of the kinase.

Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.

Amino Acid Sequence↗
PubMed2004

Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-kappaB inducing kinase NIK.

It has been shown previously that the transcription factor NF-kappaB and its inhibitor IkappaBalpha shuttle constitutively between cytosol and nucleus. Moreover, we have recently demonstrated nucleocytoplasmic shuttling of the NF-kappaB-inducing kinase NIK, a component of the NF-kappaB pathway, which is essential for lymph node development and B-cell function. Here we show that nuclear NIK also occurs in nucleoli and that this localization is mediated by a stretch of basic amino acids in the N-terminal part of the protein (R(143)-K-K-R-K-K-K(149)). This motif is necessary and sufficient for nucleolar localization of NIK, as judged by nuclear localization of mutant versions of the full-length protein and the fact that coupling of these seven amino acids to GFP also leads to accumulation in nucleoli. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) approaches, we demonstrate a dynamic distribution between nucleoli and nucleoplasm and a high mobility of NIK in both compartments. Together with the nuclear export signal in the C-terminal portion of NIK that we have also characterized in detail, the nuclear/nucleolar targeting signals of NIK mediate dynamic circulation of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartments. We demonstrate that nuclear NIK is capable of activating NF-kappaB and that this effect is diminished by nucleolar localization. Thus, subcellular distribution of NIK to different compartments might be a means of regulating the function of this kinase.

Base Sequence↗
PubMed2004

Antineuroinflammatory effect of NF-kappaB essential modifier-binding domain peptides in the adoptive transfer model of experimental allergic encephalomyelitis.

It has been shown that peptides corresponding to the NF-kappaB essential modifier-binding domain (NBD) of IkappaB kinase alpha or IkappaB kinase beta specifically inhibit the induction of NF-kappaB activation without inhibiting the basal NF-kappaB activity. The present study demonstrates the effectiveness of NBD peptides in inhibiting the disease process in adoptively transferred experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms of EAE were much lower in mice receiving wild-type (wt)NBD peptides compared with those receiving mutated (m)NBD peptides. Histological and immunocytochemical analysis showed that wtNBD peptides inhibited EAE-induced spinal cord mononuclear cell invasion and normalized p65 (the RelA subunit of NF-kappaB) expression within the spinal cord. Analysis of lymph node cells isolated from donor and recipient mice showed that wtNBD peptides but not mNBD peptides were able to shift the immune response from a Th1 to a Th2 profile. Consistently, wtNBD peptides but not mNBD peptides inhibited the encephalitogenicity of myelin basic protein-specific T cells. Furthermore, i.p. injection of wtNBD peptides but not mNBD peptides was also able to reduce LPS- and IFN-gamma-induced expression of inducible NO synthase, IL-1beta, and TNF-alpha in vivo in the cerebellum. Taken together, our results support the conclusion that NBD peptides are antineuroinflammatory, and that NBD peptides may have therapeutic effect in neuroinflammatory disorders such as multiple sclerosis.

Adoptive Transfer↗
PubMed2004

NF-kappa B activation in human breast cancer specimens and its role in cell proliferation and apoptosis.

Lack of molecular targets in estrogen receptor-negative (ER-negative) breast cancer is a major therapeutic hurdle. We studied NF-kappa B activation in human breast tumors and in carcinoma cell lines. Activated NF-kappa B was detected predominantly in ER-negative vs. ER-positive breast tumors and mostly in ER-negative and ErbB2-positive tumors (86%). These in vivo results demonstrate association of activated NF-kappa B with a subgroup of human breast tumors and are consistent with previously reported in vitro observations using similar classes of human breast cancer cell lines. Finding such an association suggested functional and biological significance. Immunofluorescence demonstrated increased nuclear p65, a component of the active NF-kappa B complex, in cytokeratin 19 (CK19)-positive epithelial cells of ER-negative/ErbB2-positive tumor samples. In contrast, nuclear NF-kappa B was detected mostly in stroma of ER-negative and ErbB2-negative tumors, suggesting a role of activated NF-kappa B in intercellular signaling between epithelial and stromal cells in this type of breast cancers. To elucidate roles of activated NF-kappa B, we used an ER-negative and ErbB2-positive human breast tumor cell line (SKBr3). The polypeptide heregulin beta1 stimulated, and herceptin, the anti-ErbB2 antibody, inhibited, NF-kappa B activation in SKBr3 cells. The NF-kappa B essential modulator (NEMO)-binding domain (NBD) peptide, an established selective inhibitor of I kappa B-kinase (IKK), blocked heregulin-mediated activation of NF-kappa B and cell proliferation, and simultaneously induced apoptosis only in proliferating and not resting cells. These results substantiate the hypothesis that certain breast cancer cells rely on NF-kappa B for aberrant cell proliferation and simultaneously avoid apoptosis, thus implicating activated NF-kappa B as a therapeutic target for distinctive subclasses of ER-negative breast cancers.

Amino Acid Sequence↗
PubMed2004

Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo.

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-kappa B (NF-kappa B) has a crucial role in osteoclast differentiation, and blocking NF-kappa B is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the I kappa B-kinase complex, a crucial component of signal transduction pathways to NF-kappa B. The peptide inhibited RANKL-stimulated NF-kappa B activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-alpha and interleukin-1 beta, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-kappa B activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption.

Animals↗
PubMed2004

A toll-like receptor that prevents infection by uropathogenic bacteria.

Toll-like receptors (TLRs) recognize molecular patterns displayed by microorganisms, and their subsequent activation leads to the transcription of appropriate host-defense genes. Here we report the cloning and characterization of a member of the mammalian TLR family, TLR11, that displays a distinct pattern of expression in macrophages and liver, kidney, and bladder epithelial cells. Cells expressing TLR11 fail to respond to known TLR ligands but instead respond specifically to uropathogenic bacteria. Mice lacking TLR11 are highly susceptible to infection of the kidneys by uropathogenic bacteria, indicating a potentially important role for TLR11 in preventing infection of internal organs of the urogenital system.

Amino Acid Sequence↗
PubMed2003

Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis.

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor beta (TGFbeta) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Tumor necrosis factor-alpha induces nuclear factor-kappaB-dependent TRPC1 expression in endothelial cells.

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.

5' Untranslated Regions↗
PubMed2003

Differential phosphorylation of the signal-responsive domain of I kappa B alpha and I kappa B beta by I kappa B kinases.

NF-kappa B activity is regulated by its association with the inhibitory I kappa B proteins, among which I kappa B alpha and I kappa B beta are the most abundant. I kappa B proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-kappa B proteins. The degradation of I kappa B proteins allows nuclear translocation of NF-kappa B and hence plays a critical role in NF-kappa B activation. Previous studies have demonstrated that, although both I kappa B proteins are phosphorylated by the same I kappa B kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate I kappa B alpha and I kappa B beta. We found that, whereas IKK alpha is a weak kinase for the N-terminal serines of both I kappa B isoforms, IKK beta is an efficient kinase for those residues in I kappa B alpha. However, IKK beta phosphorylates the N-terminal serines of I kappa B beta far less efficiently, thereby providing an explanation for the slower rate of degradation observed for I kappa B beta. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of I kappa B alpha and I kappa B beta to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of I kappa B alpha and I kappa B beta.

Animals↗
PubMed2003

X-ray crystal structure of an IkappaBbeta x NF-kappaB p65 homodimer complex.

We report the crystal structure of a murine IkappaBbeta x NF-kappaB p65 homodimer complex. Crystallographic models were determined for two triclinic crystalline systems and refined against data at 2.5 and 2.1 A. The overall complex structure is similar to that of the IkappaBalpha.NF-kappaB p50/p65 heterodimer complex. One NF-kappaB p65 subunit nuclear localization signal clearly contacts IkappaBbeta, whereas a homologous segment from the second subunit of the homodimer is mostly solvent-exposed. The unique 47-amino acid insertion between ankyrin repeats three and four of IkappaBbeta is mostly disordered in the structure. Primary sequence analysis and differences in the mode of binding at the IkappaBbeta sixth ankyrin repeat and NF-kappaB p65 homodimer suggest a model for nuclear IkappaBbeta.NF-kappaB.DNA ternary complex formation. These unique structural features of IkappaBbeta may contribute to its ability to mediate persistent NF-kappaB activation.

Binding Sites↗
PubMed2003

The interferon-inducible p202a protein modulates NF-kappaB activity by inhibiting the binding to DNA of p50/p65 heterodimers and p65 homodimers while enhancing the binding of p50 homodimers.

p202a is a member of the interferon-inducible murine p200 family of proteins. These proteins share 1 or 2 partially conserved 200 amino acid segments of the a or the b type. The known biological activities of p202a include among others the regulation of muscle differentiation, cell proliferation, and apoptosis. These biological activities of p202a can be correlated with the inhibition of the activity of several transcription factors. Thus, the binding of p202a results in the inhibition of the sequence-specific binding to DNA of the c-Fos, c-Jun, E2F1, E2F4, MyoD, myogenin, and c-Myc transcription factors. This study concerns the mechanisms by which p202a inhibits the activity of NF-kappaB, a transcription factor involved among others in host defense, inflammation, immunity, and the apoptotic response. NF-kappaB consists of p50 and p65 subunits. We demonstrate that p202a can inhibit in vitro and in vivo the binding to DNA of p65 homodimers and p50/65 heterodimers, whereas it increases the binding of p50 homodimers. Thus p202a can impair NF-kappaB activity both by inhibiting the binding to DNA of the transcriptionally active p65 homodimers and p50/p65 heterodimers and by boosting the binding of the repressive p50 homodimers. p202a can bind p50 and p65 in vitro and in vivo, and p202a can be part of the p50 homodimer complex bound to DNA. p50 binds in p202a to the a type segment, whereas p65 binds to the b type segment. Transfected ectopic p202a increases the apoptotic effect of tumor necrosis factor (at least in part) by inhibiting NF-kappaB and its antiapoptotic activity.

Animals↗
PubMed2003

RelB forms transcriptionally inactive complexes with RelA/p65.

RelB is an unusual member of the NF-kappaB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-kappaB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-kappaB1 or p52/NF-kappaB2, the precise molecular mechanisms through which it represses NF-kappaB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-alpha-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA.RelB heterodimers that were unable to bind to kappaB sites in vitro strongly suggesting that these newly described NF-kappaB dimers cannot bind DNA. Expression pattern analysis revealed that RelA.RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKbeta. Functional characterization of RelA.RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IkappaB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA.RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-kappaB-dependent gene expression.

3T3 Cells↗
PubMed2003

The MAGUK family protein CARD11 is essential for lymphocyte activation.

Members of the MAGUK family proteins cluster receptors and intracellular signaling molecules at the neuronal synapse. We report that genetic inactivation of the MAGUK family protein CARD11/Carma1/Bimp3 results in a complete block in T and B cell immunity. CARD11 is essential for antigen receptor- and PKC-mediated proliferation and cytokine production in T and B cells due to a selective defect in JNK and NFkappaB activation. Moreover, B cell proliferation and JNK activation were impaired upon stimulation of TLR4 with lipopolysaccharide, indicating that CARD11 is involved in both the innate and adaptive immune systems. Our results show that the same family of molecules are critical regulators of neuronal synapses and immune receptor signaling.

Animals↗
PubMed2002

Characterization of the Ikappa B-kinase NEMO binding domain.

Proinflammatory activation of NF-kappaB requires an upstream kinase complex (IkappaB-kinase; IKK) composed of two catalytic subunits (IKKalpha and IKKbeta) and a noncatalytic regulatory component named NEMO (NF-kappaB essential modulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKKbeta interactions and inhibits tumor necrosis factor-alpha-induced NF-kappaB. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKKalpha and IKKbeta, and mutational analysis of the IKKbeta NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKKbeta through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKKbeta NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex.

Animals↗