Inflammation in tendinopathy.

Pain and functional limitation are frequent in symptomatic tendinopathy. The essential lesion of tendinopathy is a failed healing response. Understanding the cellular and molecular mechanisms involved in a failed healing response during the early stages of pathogenesis of tendinopathy would help to develop new and effective treatments. The role of inflammation in the development of tendon pathologies has been revived during the last few years, in particular during the first phases of tendinopathies, when "early tendinopathy" may not be clinically evident. This review outlines the possible molecular events that occur in the first phases of tendinopathy onset, stressing the role of pro-inflammatory cytokines, proteolytic enzymes, growth factors and healing genes in the development of tendon disorders.

The neutrophil gelatinase-associated lipocalin (NGAL), a NF-kappaB-regulated gene, is a survival factor for thyroid neoplastic cells.

NF-kappaB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-kappaB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-kappaB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-kappaB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-kappaB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-kappaB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with alpha-enolase devoid of plasminogen binding activity.

HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.

Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-kappaB activation.

The molecular complex containing BCL10 and CARMA [CARD (caspase recruitment domain)-containing MAGUK (membrane-associated guanylate kinase)] proteins has recently been identified as a key component in the signal transduction pathways that regulate activation of the transcription factor NF-kappaB (nuclear factor kappaB) in lymphoid and non-lymphoid cells. Assembly of complexes containing BCL10 and CARMA proteins relies on homophilic interactions established between the CARDs of these proteins. In order to identify BCL10-inhibitory peptides, we have established a method of assaying peptides derived from the CARD of BCL10 in binding competition assays of CARD-CARD self-association. By this procedure, a short peptide corresponding to amino acid residues 91-98 of BCL10 has been selected as an effective inhibitor of protein self-association. When tested in cell assays for its capacity to block NF-kappaB activation, this peptide represses activation of NF-kappaB mediated by BCL10, CARMA3 and PMA/ionomycin stimulation. Collectively, these results indicate that residues 91-98 of BCL10 are involved in BCL10 self-association and also participate in the interaction with external partners. We also show that blocking of the CARD of BCL10 may potentially be used for the treatment of pathological conditions associated with inappropriate NF-kappaB activation.

Conformational stability and DNA binding energetics of the rat thyroid transcription factor 1 homeodomain.

The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF-1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF-1HD is a reversible two-state transition. The protein is not stable against temperature, showing a denaturation temperature of 32 degrees C in the absence of salt and 50 degrees C at 75 mM KCl. The binding energetics of TTF-1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25 degrees C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 x 10(8)M(-1) and the binding enthalpy change amounts to -41 kJ mol(-1). The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF-1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF-1HD with the target DNA sequence and TTF-1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK-2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF-1HD.

RbAp48 is a target of nuclear factor-kappaB activity in thyroid cancer.

CONTEXT: We have recently shown that nuclear factor (NF)-kappaB activity is constitutively elevated in anaplastic human thyroid carcinomas. The inhibition of NF-kappaB in the anaplastic thyroid carcinoma cell line (FRO) leads to increased susceptibility to apoptosis induced by chemotherapeutic drugs and to the block of oncogenic activity. OBJECTIVES: To understand better the molecular mechanisms played by NF-kappaB in thyroid oncogenesis, we performed a differential proteomic analysis between FRO transfected with a superrepressor form of inhibitor of kappaBalpha (IkappaBalphaM) and the parental counterpart (FRO Neo cells). RESULTS: Differential proteomic analysis revealed that the retinoblastoma-associated protein 48 (RbAp48) is down-regulated in the absence of functional NF-kappaB. Immunohistochemical analysis of normal and pathological human thyroid specimens confirmed that RbAp48 is strongly overexpressed in primary human carcinomas. Reduction of RbAp48 expression using small interfering RNA determined the suppression of tumorigenicity, very likely due to the decrease of their growth rate rather than to an increased susceptibility to apoptosis. In addition, we showed that NF-kappaB, at least in part, transcriptionally controls RbAp 48. A functional NF-kappaB consensus sequence was located within the promoter region of RbAp48 human gene, and embryonic fibroblasts isolated from the p65 knockout mouse (murine embryonic fibroblasts p65-/-) showed decreased expression of RbAp48. CONCLUSION: Our results show that RbAp48 is a NF-kappaB-regulated gene playing an important role in thyroid cancer cell autonomous proliferation.

Oligosaccharidic fractions derived from Triticum vulgare extract accelerate tissutal repairing processes in in vitro and in vivo models of skin lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The aqueous extract of Triticum vulgare (TVE) is currently an active component used by Farmaceutici Damor in the manufacture of certain pharmaceutical products already marketed in Italy and abroad under the brand name Fitostimoline(®), in the formulation of cream and medicated gauze and is commonly used for the treatment of decubitus ulcers, sores, burns, scarring delays, dystrophic diseases, and, more broadly, in the presence of problems relating to re-epithelialization or tissue regeneration. The active components of Fitostimoline(®)-based products determine a marked acceleration of tissutal repairing processes, stimulate chemotaxis and the fibroblastic maturation, and significantly increase the fibroblastic index, which are crucial points in the repairing processes. The aim of the present paper was to identify and characterize the active fractions of TVE responsible for the pharmacological effect in tissutal repairing processes. MATERIALS AND METHODS: Several fractions obtained from TVE by ultrafiltration procedures and HPAE chromatography were tested to measure their growth-enhancing activity on NIH-3T3 fibroblasts. The healing action of the same fractions, prepared as cream formulation, was assessed in rat subjected to two different models of skin lesion, skin scarification and excision. RESULTS: Our results showed a pro-proliferative effect of the fractions ST-98 and K>1000 in NIH-3T3 fibroblasts. Moreover these fractions formulated as cream preparations were effective also in in vivo models of skin lesion. CONCLUSIONS: The results of the present study showed that these active fractions of TVE are responsible for its pro-proliferative effect.

Endoplasmic reticulum stress causes thyroglobulin retention in this organelle and triggers activation of nuclear factor-kappa B via tumor necrosis factor receptor-associated factor 2.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

Promoter identification of CIKS, a novel NF-kappaB activating gene, and regulation of its expression.

We have recently identified a novel gene, named CIKS (Connection to IKK-complex and SAPK), able to activate the transcription factor NF-kappaB, after interaction with the regulatory subunit NEMO/IKKgamma of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5'-flanking region of CIKS gene was 'TATA-less', but contained other consensus promoter elements including an initiator (Inr), 'GC' and 'CAAT' boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5'-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from -464 to -220 bp of the 5'-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase-PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and transforming growth factor (TGF)-beta, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5'-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.

TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2+-ATPases expression and activity in PC Cl3 thyroid cells.

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.

Nuclear factor-{kappa}B contributes to anaplastic thyroid carcinomas through up-regulation of miR-146a.

CONTEXT: Micro-RNAs (miRNAs) have been recently involved in the modulation of several biological activities including cancer. Many human tumors show deregulated expression of miRNAs targeting oncogenes and/or tumor suppressors, thus identifying miRNAs as new molecular targets for cancer therapy. OBJECTIVES: Nuclear factor (NF)-kappaB is strongly activated in human anaplastic thyroid carcinomas (ATCs). Because the regulation of miRNA expression is under control of RNA polymerase II-dependent transcription factors, we stably inactivated NF-kappaB in the ATC-derived FRO cell line and analyzed its miRNA profile in comparison with the parental counterpart by using a miRNA chip microarray. RESULTS: The analysis revealed that a number of miRNAs were differentially expressed in the two cell lines. Among others, the miR-146a showed a strong down-regulation that was confirmed by quantitative real time RT-PCR. The expression of miR-146a was almost undetectable in mouse embryonic fibroblasts isolated from the RelA knockout mice and was restored after reexpression of RelA, thus indicating that miR-146a transcription was controlled by NF-kappaB. The inhibition of miR-146a expression in FRO cells decreased their oncogenic potential and increased the susceptibility to chemotherapeutic drug-induced apoptosis. No difference was found in the growth rate between untransfected and miR-146a-null FRO cells. Importantly, the miR-146a resulted in overexpression of human ATC specimens compared with the normal thyroid tissue. CONCLUSIONS: Our results show that NF-kappaB contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a.

TNF receptor-associated factor 1 is a positive regulator of the NF-kappaB alternative pathway.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is unique among the members of the TRAF family, as it lacks the N-terminal RING/zinc-finger domain. Also the function of TRAF1 is not clearly established, with many papers reporting contradictory results. Here we show that TRAF1 interacts with BAFF receptor, a member of the TNF receptor family, and positively regulates activation of the alternative NF-kappaB pathway. Ectopic expression of TRAF1 causes degradation of TRAF3, stabilization of NIK, and processing of p100 to produce the mature form p52. In addition, we show that knocking-down expression of TRAF1 in the Hodgkin's disease derived cell line L1236, interfere with p100 processing and with p52 mediate gene transcription. Collectively these results support a role for TRAF1 as a positive regulator of the NF-kappaB alternative pathway.

ABIN-1 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB.

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de-ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB.

A single chondroitin 6-sulfate oligosaccharide unit at Ser-2730 of human thyroglobulin enhances hormone formation and limits proteolytic accessibility at the carboxyl terminus. Potential insights into thyroid homeostasis and autoimmunity.

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 '-triiodothyronine (T3) (171%) and 3,5,3',5'-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.

Central role of the scaffold protein tumor necrosis factor receptor-associated factor 2 in regulating endoplasmic reticulum stress-induced apoptosis.

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-kappaB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-kappaB in protecting cells from ER stress-induced apoptosis.

Thyroglobulin as an autoantigen: what can we learn about immunopathogenicity from the correlation of antigenic properties with protein structure?

Autoantibodies against human thyroglobulin are a hallmark of autoimmune thyroid disease in humans, and are often found in normal subjects. Their pathogenic significance is debated. Several B-cell epitope-bearing peptides have been identified in thyroglobulin. They are generally located away from the cysteine-rich regions of tandem sequence repetition. It is possible that our current epitopic map is incomplete because of the difficulty that proteolytic and recombinant approaches have in restituting conformational epitopes based upon proper pairing between numerous cysteinyl residues. Furthermore, the homology of cysteine-rich repeats with a motif occurring in several proteins, endowed with antiprotease activity, suggests that these regions may normally escape processing and presentation to the immune system, and brings attention to the mechanisms, such as oxidative cleavage, by which such cryptic epitopes may be exposed. A number of T-cell epitope-bearing peptides, endowed with thyroiditogenic power in susceptible mice, were also identified. None of them was dominant, as none was able to prime in vivo lymph node cells that would proliferate or transfer autoimmune thyroiditis to syngeneic hosts, upon stimulation with intact thyroglobulin in vitro. More than half of them are located within the acetylcholinesterase-homologous domain of thyroglobulin, and overlap B-cell epitopes associated with autoimmune thyroid disease, while the others are located within cysteine-rich repeats. The immunopathogenic, non-dominant character of these epitopes also favours the view that the development of autoimmune thyroid disease may involve the unmasking of cryptic epitopes, whose exposure may cause the breaking of peripheral tolerance to thyroglobulin. Further research in this direction seems warranted.

SDS-resistant active and thermostable dimers are obtained from the dissociation of homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus in SDS. Stabilizing role of the A-C intermonomeric interface.

beta-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus (M(r) 226,760) has shown that addition of 0.05-0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on beta-glycosidase quaternary structure. In 0.1-1% SDS, the enzyme was dimeric, as determined by Ferguson analysis of transverse-gradient polyacrylamide gels. The catalytic activity of the beta-glycosidase dimer in SDS was determined by in-gel assay. A minor decrease of thermal stability in SDS was observed after exposure to temperatures up to 80 degrees C for 1 h. An analysis of beta-glycosidase crystal structure showed different changes in solvent-accessible surface area on going from the tetramer to the two possible dimers (A-C and A-D). Energy minimization and molecular dynamics calculations showed that the A-C dimer, exhibiting the lowest exposed surface area, was more stabilized by a network of polar interactions. The charge distribution around the A-C interface was characterized by a local short range anisotropy, resulting in an unfavorable interaction with SDS. This paper provides a detailed description of an SDS-resistant inter-monomeric interface, which may help understand similar interfaces involved in important biological processes.

Role of the adaptor protein CIKS in the activation of the IKK complex.

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in numerous cellular processes, including stress response, inflammation, and protection from apoptosis. Therefore, the activity of NF-kappaB needs to be tightly regulated. We have previously identified a novel gene, named CIKS (connection to IkappaB-kinase and SAPK), able to bind the regulatory sub-unit NEMO/IKKgamma and to activate NF-kappaB. Here, we demonstrate that CIKS forms homo-oligomers, interacts with NEMO/IKKgamma, and is recruited to the IKK-complex upon cell stimulation. In addition, we identified the regions of CIKS responsible for these functions. We found that the ability of CIKS to oligomerize, and to be recruited to the IKK-complex is not sufficient to activate the NF-kappaB. In fact, a deletion mutant of CIKS able to oligomerize, to interact with NEMO/IKKgamma, and to be recruited to the IKK-complex does not activate NF-kappaB, suggesting that CIKS needs a second level of regulation to efficiently activate NF-kappaB.

Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca(2+) from the ER elicited by thapsigargin (a specific inhibitor of ER Ca(2+)-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca(2+) strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

Physical and functional interaction of CARMA1 and CARMA3 with Ikappa kinase gamma-NFkappaB essential modulator.

CARMA proteins are scaffold molecules that contain a caspase recruitment domain and a membrane-associated guanylate kinase-like domain. CARMA1 plays a critical role in mediating activation of the NFkappaB transcription factor following antigen receptor stimulation of both B and T lymphocytes. However, the biochemical mechanism by which CARMA1 regulates activation of NFkappaB remains to be determined. Here we have shown that CARMA1 and CARMA3 physically associate with Ikappa kinase gamma/NFkappaB essential modulator (IkappaKgamma-NEMO) in lymphoid and non-lymphoid cells. CARMA1 participates to an inducible large molecular complex that contains IkappaKgamma/NEMO, Bcl10, and IkappaKalpha/beta kinases. Expression of the NEMO-binding region of CARMA3 exerts a dominant negative effect on Bcl10-mediated activation of NFkappaB. Thus, our results provide direct evidence for physical and functional interaction between CARMA and the IkappaK complex and offer a biochemical framework to understand the molecular activities controlled by CARMA-1, -2, and -3 and Bcl10.