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Biomedical subjects

Sankar Ghosh

Publications and source records attributed to Sankar Ghosh.

At least 19 recordsLinked to original sources
PubMed2026

An inflammatory bowel disease-linked lncRNA suppresses transcription factor T-BET expression in T cells to limit intestinal inflammation.

Among the tens of thousands of annotated long noncoding RNAs (lncRNAs) in the human genome, only a small fraction have been functionally characterized. Here, we show that a well-established inflammatory bowel disease (IBD) risk locus encoded a conserved lncRNA, lnc15 (2310015A10Rik/ENSMUSG00000097729), whose structure was destabilized by risk-associated variants, leading to its degradation. Deletion of lnc15 in mice resulted in molecular features of inflammation under steady-state conditions and conferred heightened susceptibility to experimental colitis. Lnc15 was abundantly expressed in T cells, with highest expression in regulatory T (Treg) cells. Mechanistically, lnc15 suppressed the transcription factor T-BET by recruiting the CCR4-NOT RNA degradation complex to Tbx21 mRNA. Our study identifies that lnc15 simultaneously enhances Treg cell suppressive function and impairs conventional T cell pathogenicity in the context of intestinal inflammation. Collectively, these findings identify lnc15 as a functional lncRNA that links noncoding genetic variation to immune regulation and prevention of mucosal inflammation. VIDEO ABSTRACT.

RNA, Long Noncoding↗
PubMed2006

Dimerization of the I kappa B kinase-binding domain of NEMO is required for tumor necrosis factor alpha-induced NF-kappa B activity.

Previous studies have demonstrated that peptides corresponding to a six-amino-acid NEMO-binding domain from the C terminus of IkappaB kinase alpha (IKKalpha) and IKKbeta can disrupt the IKK complex and block NF-kappaB activation. We have now mapped and characterized the corresponding amino-terminal IKK-binding domain (IBD) of NEMO. Peptides corresponding to the IBD were efficiently recruited to the IKK complex but displayed only a weak inhibitory potential on cytokine-induced NF-kappaB activity. This is most likely due to the formation of sodium dodecyl sulfate- and urea-resistant NEMO dimers through a dimerization domain at the amino terminus of NEMO that overlaps with the region responsible for binding to IKKs. Mutational analysis revealed different alpha-helical subdomains within an amino-terminal coiled-coil region are important for NEMO dimerization and IKKbeta binding. Furthermore, NEMO dimerization is required for the tumor necrosis factor alpha-induced NF-kappaB activation, even when interaction with the IKKs is unaffected. Hence, our data provide novel insights into the role of the amino terminus of NEMO for the architecture of the IKK complex and its activation.

Animals↗
PubMed2006

NF-kappaB is dispensable for normal lymphocyte development in bone marrow but required for protection of progenitors from TNFalpha.

Levels of the nuclear factor-kappa B (NF-kappaB)/Rel family of proteins are carefully modulated in differentiating lymphocytes, where these transcription factors are thought to be important for survival and fate decisions. In contrast, gene-targeting experiments have not revealed clear roles for these transcription factors in lymphopoiesis within bone marrow. Inhibition of NF-kappaB by introduction of mutated I kappa B alpha, a 'superinhibitor' of NF-kappaB, into hematopoietic stem cells or early progenitors suppressed B as well as T lymphopoiesis following transplantation into immunodeficient mice. Furthermore, a NF-kappaB essential modifier-binding domain (NBD) peptide that blocks IKB kinase (IKK) activity selectively impaired the generation of adult B lineage cells. However, this suppression did not occur when a neutralizing antibody to tumor necrosis factor alpha (TNFalpha) was added to the cultures, or in circumstances where few non-lymphoid cells were present. We conclude that while NF-kappaB plays a survival-promoting role in lymphoid progenitors, this may only be significant in circumstances such as transplantation when levels of TNFalpha are high.

Adoptive Transfer↗
PubMed2006

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

Activin Receptors, Type I↗
PubMed2006

Antigen-receptor signaling to nuclear factor kappa B.

Signal transduction events leading to the survival, differentiation, or apoptosis of cells of the innate or adaptive immune system must be properly coordinated to ensure the normal mounting and termination of immune responses. One of the key transcription factors in immune responses is nuclear factor kappaB (NF-kappaB), which has been the focus of intense investigation over the past two decades. With the identification of the CARMA1-BCL10-MALT1 complex and ongoing progress in understanding the molecular mechanisms connecting T cell and B cell receptor proximal signals to the IkappaB kinase (IKK) complex, a cohesive model of antigen receptor (AgR)-dependent signaling to NF-kappaB activation is beginning to emerge. In this review, we provide an overview of the current state of research into the mechanisms that regulate AgR-mediated NF-kappaB transcriptional activity, with particular focus on the events leading to activation of the IKK complex.

Animals↗
PubMed2006

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor.

CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-kappaB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-kappaB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-kappaB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein kappaB kinase (IKK) activation, a major upstream kinase mediating NF-kappaB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-kappaB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.

Animals↗
PubMed2006

Recognition and signaling by toll-like receptors.

Toll-like receptors (TLRs) are transmembrane proteins that detect invading pathogens by binding conserved, microbially derived molecules and that induce signaling cascades for proinflammatory gene expression. A critical component of the innate immune system, TLRs utilize leucine-rich-repeat motifs for ligand binding and a shared cytoplasmic domain to recruit the adaptors MyD88, TRIF, TIRAP, and/or TRAM for downstream signaling. Despite significant domain conservation, TLRs induce gene programs that lead not only to the robust production of general proinflammatory mediators but also to the production of unique effectors, which provide pathogen-tailored immune responses. Here we review the mechanisms by which TLRs recognize pathogens and induce distinct signaling cascades.

Animals↗
PubMed2006

Local treatment with the selective IkappaB kinase beta inhibitor NEMO-binding domain peptide ameliorates synovial inflammation.

Nuclear factor (NF)-kappaB is a key regulator of synovial inflammation. We investigated the effect of local NF-kappaB inhibition in rat adjuvant arthritis (AA), using the specific IkappaB kinase (IKK)-beta blocking NF-kappaB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-beta-induced IkappaB alpha phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-alpha and IL-1-beta in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-beta-induced TNF-alpha production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-alpha-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-kappaB blockade using a small peptide inhibitor of IKK-beta has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-beta-targeted NF-kappaB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.

Animals↗
PubMed2005

TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo.

TGF-beta-activated kinase 1 (TAK1), a member of the MAPKKK family, is thought to be a key modulator of the inducible transcription factors NF-kappaB and AP-1 and, therefore, plays a crucial role in regulating the genes that mediate inflammation. Although in vitro biochemical studies have revealed the existence of a TAK1 complex, which includes TAK1 and the adapter proteins TAB1 and TAB2, it remains unclear which members of this complex are essential for signaling. To analyze the function of TAK1 in vivo, we have deleted the Tak1 gene in mice, with the resulting phenotype being early embryonic lethality. Using embryonic fibroblasts lacking TAK1, TAB1, or TAB2, we have found that TNFR1, IL-1R, TLR3, and TLR4-mediated NF-kappaB and AP-1 activation are severely impaired in Tak1(m/m) cells, but they are normal in Tab1(-/-) and Tab2(-/-) cells. In addition, Tak1(m/m) cells are highly sensitive to TNF-induced apoptosis. TAK1 mediates IKK activation in TNF-alpha and IL-1 signaling pathways, where it functions downstream of RIP1-TRAF2 and MyD88-IRAK1-TRAF6, respectively. However, TAK1 is not required for NF-kappaB activation through the alternative pathway following LT-beta signaling. In the TGF-beta signaling pathway, TAK1 deletion leads to impaired NF-kappaB and c-Jun N-terminal kinase (JNK) activation without impacting Smad2 activation or TGF-beta-induced gene expression. Therefore, our studies suggests that TAK1 acts as an upstream activating kinase for IKKbeta and JNK, but not IKKalpha, revealing an unexpectedly specific role of TAK1 in inflammatory signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Activation of NF-kappaB promotes the transition of large, CD43+ pre-B cells to small, CD43- pre-B cells.

The regulation of the transcription factor nuclear factor-kappaB (NF-kappaB) during B-cell development was examined using cells isolated from the bone marrow of transgenic mice expressing a kappaB luciferase reporter gene. The results indicate that the highest level of NF-kappaB activity is present in cells expressing the pre-B-cell receptor. Furthermore, cross-linking of Igbeta on CD43(+) pre-B cells is able to activate NF-kappaB in recombination-activating gene 1-deficient mice, preceding their further differentiation into CD43(-) pre-B cells. Expression of a dominant negative form of IkappaBalpha using a transgenic approach or by retroviral infection leads to a reduction in the number of CD43(+) pre-B cells. These data therefore indicate that activation of NF-kappaB in CD43(+) pre-B cells, as a result of signaling by the pre-B-cell receptor, facilitates the continued development of large, CD43(+) pre-B cells into small CD43(-) pre-B cells.

Animals↗
PubMed2005

Regulating inducible transcription through controlled localization.

Many signaling pathways regulate the activity of effector transcription factors by controlling their subcellular localization. Until recently, the cytoplasmic retention of inactive transcription factors was mainly attributed to binding partners that mask the nuclear localization signals (NLSs) of target proteins. Inactive transcription factors were thought to be exclusively cytoplasmic until their activation, after which the NLSs were unmasked to allow nuclear translocation. There is now a growing body of evidence, however, that challenges this simple model. This review discusses recent reports that suggest that inducible transcription factors can constantly shuttle between the cytoplasm and the nucleus, and that their apparent cytoplasmic retention can be achieved by binding partners that mask the NLSs, tether the transcription factor to cytoplasmic structures, or mark the transcription factor for proteasomal degradation. We also discuss the possibility that this more complex model of cytoplasmic retention might be applicable to a broader range of transcription factors and their associated signaling pathways.

Animals↗
PubMed2005

TLR11 activation of dendritic cells by a protozoan profilin-like protein.

Mammalian Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs). Although TLRs are clearly involved in the detection of bacteria and viruses, relatively little is known about their function in the innate response to eukaryotic microorganisms. Here we identify a profilin-like molecule from the protozoan parasite Toxoplasma gondii that generates a potent interleukin-12 (IL-12) response in murine DCs that is dependent on myeloid differentiation factor 88. T. gondii profilin activates DCs through TLR11 and is the first chemically defined ligand for this TLR. Moreover, TLR11 is required in vivo for parasite-induced IL-12 production and optimal resistance to infection, thereby establishing a role for the receptor in host recognition of protozoan pathogens.

Adaptor Proteins, Signal Transducing↗
PubMed2005

PDK1 nucleates T cell receptor-induced signaling complex for NF-kappaB activation.

Activation of the transcription factor NF-kappaB after engagement of the T cell receptor (TCR) is important for T cell proliferation and activation during the adaptive immune response. Recent reports have elucidated a signaling pathway that involves the protein kinase C (PKC), the scaffold protein CARD11 (also called CARMA-1), the caspase recruitment domain (CARD)-containing protein Bcl10, and the paracaspase (protease related to caspases) MALT1 as critical intermediates linking the TCR to the IkappaB kinase (IKK) complex. However, the events proximal to the TCR that initiate the activation of this signaling pathway remain poorly defined. We demonstrate that 3-phosphoinositide-dependent kinase 1 (PDK1) has an essential role in this pathway by regulating the activation of PKC and through signal-dependent recruiting of both PKC and CARD11 to lipid rafts. PDK1-associated PKC recruits the IKK complex, whereas PDK1-associated CARD11 recruits the Bcl10-MALT1 complex, thereby allowing activation of the IKK complex through Bcl10-MALT1-dependent ubiquitination of the IKK complex subunit known as NEMO (NF-kappaB essential modifier). Hence, PDK1 plays a critical role by nucleating the TCR-induced NF-kappaB activation pathway in T cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2005

Amelioration of acute inflammation by systemic administration of a cell-permeable peptide inhibitor of NF-kappaB activation.

OBJECTIVE: We used an experimental model of inflammation in mice, carrageenan-induced paw edema, to study the antiinflammatory effects of the NEMO-binding domain (NBD) peptide, which blocks activation of the inducible transcription factor NF-kappaB. METHODS: Paw edema was induced by subplantar injection of 1% lambda-carrageenan into the mouse left hind paw. Test agents were given intraperitoneally immediately after carrageenan injection. The increase in footpad thickness was considered to be edema. In some experiments, the mice were killed and the paws were removed for histologic and molecular biology analysis. NF-kappaB DNA binding activity was evaluated in nuclear extracts by electrophoretic mobility shift assays. The expression levels of NF-kappaB-regulated cyclooxygenase 2 (COX-2) protein and tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) were evaluated by immunoblot analysis and polymerase chain reaction amplification of reverse-transcribed mRNA, respectively. RESULTS: We found that systemically administered NBD peptide significantly inhibited edema formation and cellular infiltration in inflamed mouse paws. This antiinflammatory activity was most likely due to inhibition of expression of proinflammatory mediators, such as TNFalpha and COX-2, in inflamed tissues. CONCLUSION: These studies further establish NF-kappaB as a target for antiinflammatory therapy and provide support for the use of the NBD peptide as a possible therapeutic agent for inflammatory diseases.

Animals↗
PubMed2005

Selective inhibition of NF-kappaB in dendritic cells by the NEMO-binding domain peptide blocks maturation and prevents T cell proliferation and polarization.

Dendritic cells (DC) are the only antigen-presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF-kappaB dependent, we hypothesized that blocking NF-kappaB activity in DC by selectively targeting the inhibitor of kappaB (IkappaB) kinase (IKK) complex using the novel NF-kappaB inhibitor NEMO-binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte-derived DC to test the biological effects of the NBD peptide in vitro. NF-kappaB inhibition by the NBD peptide resulted in blockade of IKK-mediated IkappaBalpha phosphorylation and subsequent nuclear translocation and DNA binding of NF-kappaB p65 in DC. In addition, IL-6, IL-12, and TNF-alpha production was dose-dependently blocked and NBD peptide treatment also led to a strong reduction of LPS-induced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF-kappaB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses.

Cell Differentiation↗