Mad2 Research Articles

Alcohol exposure regulates heat shock transcription factor binding and heat shock proteins 70 and 90 in monocytes and macrophages: implication for TNF-alpha regulation.

Immunomodulatory effects of alcohol use involve regulation of innate immune cell function leading to liver disease. Alteration of inflammatory responses by alcohol is linked to dysregulated TNF-alpha production. Alcohol-induced oxidative stress also contributes to alterations in inflammatory cell activity. Heat shock proteins (hsps) and the heat shock transcription factor-1 (HSF-1) induced by oxidative stress regulate NF-kappaB activation and TNF-alpha gene expression in monocytes and macrophages. Here, we report that in vitro alcohol treatment induced and augmented LPS-induced HSF-1 nuclear translocation and DNA-binding activity in monocytes and macrophages. Supershift analysis revealed that alcohol regulated HSF-1- and not HSF-2-binding activity. Hsp70, a target gene induced by HSF-1, was transiently increased within 24 h by alcohol, but extended alcohol exposure decreased hsp70 in macrophages. The alcohol-induced alteration of hsp70 correlated with a concomitant change in hsp70 promoter activity. Hsp90, another HSF-1 target gene, was decreased during short-term alcohol but increased after prolonged alcohol exposure. Decreased hsp90-HSF-1 complexes after short-term alcohol indicated dissociation of HSF-1 from hsp90. On the other hand, hsp90 interacted with client protein IkappaB kinase beta, a signaling intermediate of the LPS pathway, followed by IkappaBalpha degradation and increased NF-kappaB activity after chronic alcohol exposure, indicating that hsp90 plays an important role in supporting inflammatory cytokine production. Inhibition of hsp90 using geldanamycin prevented prolonged alcohol-induced elevation in LPS-induced NF-kappaB and TNF-alpha production. These results suggest that alcohol exposure differentially regulates hsp70 and hsp90 via HSF-1 activation. Further, hsp90 regulates TNF-alpha production in macrophages contributing to alcohol-induced inflammation.

Mitochondria, oxygen sensing, and the regulation of HIF-2α. Focus on “Induction of HIF-2α is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line”

oxygen gradients in tissues and cells are critical signals in a range of physiological processes including development, wound healing, and adaptation to hypoxia ([12][1]). Eukaryotic cells have evolved mechanisms for sensing changes in local oxygen tension and initiation of a response designed to

Stabilization of RelB Requires Multidomain Interactions with p100/p52

The NF-kappaB family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IkappaB proteins. We show that the protein level of RelB is significantly reduced in the absence of p100 and reduced even more when both p100 and p105 are absent. RelB stabilizes itself by directly interacting with p100, p105, and their processed products. However, RelB forms complexes with its partners using different interaction modes. Although the C-terminal ankyrin repeat domain of p105 is not involved in the RelB-p105 complex formation, all domains and flexible regions of each protein are engaged in the RelB-p100 complex. In several respects the RelB-p52 and RelB-p100 complexes are unique in the NF-kappaB family. The N-terminal domain of p100/p52 interacts with RelB but not RelA. The transcriptional activation domain of RelB, but not RelA, directly interacts with the processing region of p100. These unique protein-protein contacts explain why RelB prefers p52 as its dimeric partner for transcriptional activity and is retained in the cytoplasm as an inhibited complex by p100. This association-mediated stabilization of RelB implies a possible role for RelB in the processing of p100 into p52.

NF-κB dictates the degradation pathway of IκBα

IkappaB proteins are known as the regulators of NF-kappaB activity. They bind tightly to NF-kappaB dimers, until stimulus-responsive N-terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IkappaBalpha is an unstable protein whose rapid degradation is slowed upon binding to NF-kappaB, but it is not known what dynamic mechanisms control the steady-state level of total IkappaBalpha. Here, we show clearly that two degradation pathways control the level of IkappaBalpha. Free IkappaBalpha degradation is not controlled by IKK or ubiquitination but intrinsically, by the C-terminal sequence known as the PEST domain. NF-kappaB binding to IkappaBalpha masks the PEST domain from proteasomal recognition, precluding ubiquitin-independent degradation; bound IkappaBalpha then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IkappaBalpha protein; alteration of free IkappaBalpha degradation dampens NF-kappaB activation. In addition, we find that both free and bound IkappaBalpha are similar substrates for IKK, and the preferential phosphorylation of NF-kappaB-bound IkappaBalpha is due to stabilization of IkappaBalpha by NF-kappaB.

Protective Effects of Aspirin Against Oxidized LDL-induced Inflammatory Protein Expression in Human Endothelial Cells

Atherosclerosis is a complex vascular inflammatory disease. Oxidized low-density lipoprotein (ox-LDL) is directly associated with chronic vascular inflammation. In this study, we hypothesized that nonselective cyclooxygenase inhibitor aspirin might affect the ox-LDL-induced inflammatory responses on human endothelial cells. To test this assumption, cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression, IkappaB and p38 mitogen-activated protein kinase (MAPK) phosphorylation were determined in endothelial cells exposed to ox-LDL in the presence of aspirin. The results showed that aspirin significantly suppressed COX-2 and ICAM-1 expression induced by ox-LDL and also inhibited IkappaB phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, aspirin reduced the level of p38 MAPK phosphorylation. Our findings suggest that aspirin can decrease inflammatory responses induced by ox-LDL, and the mechanism might be associated with NF-kappaB activation pathway and inhibition of p38 MAPK phosphorylation.

Role of nuclear IκB proteins in the regulation of host immune responses

A variety of microbial components activate a transcription factor called nuclear factor-kappaB (NF-kappaB) that plays an essential role in the optimal activation of host immune systems. The transcriptional activity of NF-kappaB is tightly regulated at multiple steps in immune signaling pathways, because excessive activation is detrimental to the host. One mechanism to prevent NF-kappaB activation is mediated by cytoplasmic IkappaB family proteins. Although cytoplasmic IkappaBs interact with NF-kappaB subunits in the cytoplasm of unstimulated cells, IkappaBs are rapidly degraded on stimulation, allowing free NF-kappaB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. After the translocation of NF-kappaB from the cytoplasm to the nucleus, nuclear proteins that are structurally similar to cytoplasmic IkappaBs take part in the regulation of NF-kappaB transcriptional activity, as activators or inhibitors, by associating with NF-kappaB subunits. Therefore, the regulatory IkappaB-like nuclear molecules are described as "nuclear IkappaB proteins." In this review, the in vivo function of the nuclear IkappaB proteins, Bcl-3, IkappaBzeta, and IkappaBNS in the context of host immune responses and diseases will be discussed.

Salicylate prevents hepatic insulin resistance caused by short-term elevation of free fatty acids in vivo

Recent evidence indicates that inflammatory pathways are causally involved in insulin resistance. In particular, Ikappa Balpha kinase beta (IKKbeta ), which can impair insulin signaling directly via serine phosphorylation of insulin receptor substrates (IRS) and/or indirectly via induction of transcription of proinflammatory mediators, has been implicated in free fatty acid (FFA)-induced insulin resistance in skeletal muscle. However, it is unclear whether liver IKKbeta activation plays a causal role in hepatic insulin resistance caused by acutely elevated FFA. In the present study, we wished to test the hypothesis that sodium salicylate, which inhibits IKKbeta , prevents hepatic insulin resistance caused by short-term elevation of FFA. To do this, overnight-fasted Wistar rats were subject to 7-h i.v. infusion of either saline or Intralipid plus 20 U/ml heparin (IH; triglyceride emulsion that elevates FFA levels in vivo) with or without salicylate. Hyperinsulinemic-euglycemic clamp with tracer infusion was performed to assess insulin-induced stimulation of peripheral glucose utilization and suppression of endogenous glucose production (EGP). Infusion of IH markedly decreased (P < 0.05) insulin-induced stimulation of peripheral glucose utilization and suppression of EGP, which were completely prevented by salicylate co-infusion. Furthermore, salicylate reversed IH-induced 1) decrease in Ikappa Balpha content; 2) increase in serine phosphorylation of IRS-1 (Ser 307) and IRS-2 (Ser 233); 3) decrease in tyrosine phosphorylation of IRS-1 and IRS-2; and 4) decrease in serine 473-phosphorylated Akt in the liver. These results demonstrate that inhibition of IKKbeta prevents FFA-induced impairment of hepatic insulin signaling, thus implicating IKKbeta as a causal mediator of hepatic insulin resistance caused by acutely elevated plasma FFA.

Effect of Thiol‐Containing Molecule Cysteamine on the Induction of Inducible Nitric Oxide Synthase in Hepatocytes

Cysteamine, which is a known antioxidant and anti-inflammatory agent, is believed to be a key regulator of essential metabolic pathways in organisms. Cysteamine has beneficial effects in liver damaged by a variety of insults. During liver injury, inducible nitric oxide synthase (iNOS) is induced by lipopolysaccharide or proinflammatory cytokines, leading to excessive nitric oxide (NO) production. Accumulated evidence indicates that NO is an important factor associated with hepatic dysfunction. We examined whether cysteamine influences the induction of iNOS in hepatocytes. Primary cultured rat hepatocytes were treated with interleukin (IL)-1beta in the presence and absence of cysteamine. NO production, iNOS induction, and iNOS signal were analyzed. IL-1beta stimulated the inhibitory protein kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) pathway, resulting in the activation of NFkappaB (nuclear translocation and DNA binding), which was followed by the induction of iNOS and NO production. The addition of IL-1beta and cysteamine (1-4 mmol/L) markedly inhibited NO production, with a maximal effect at 4 mmol/L (80%-90% inhibition). Cysteamine also decreased the levels of iNOS protein and mRNA. Transfection experiments revealed that cysteamine decreased the transactivation activity of the iNOS promoter. An electrophoretic mobility shift assay demonstrated that cysteamine inhibited the activation of NFkappaB. Furthermore, cysteamine decreased the mRNA levels of the NFkappaB subunit p65 but increased those of the inhibitory protein IkappaB. These findings suggest that cysteamine inhibits iNOS induction at the step of NFkappaB activation. Further study is necessary to define the molecular basis of this effect of cysteamine on the regulation of NFkappaB and its pharmacologic implications.

Doxorubicin Enhances the Expression of Transgene Under Control of the CMV Promoter in Anaplastic Thyroid Carcinoma Cells

We investigated the effect of doxorubicin on transgene expression and evaluated the mechanism of enhanced transgene expression by doxorubicin in transfected human anaplastic thyroid cancer cells (ARO cells). ARO cells were transfected with plasmid vectors or adenoviral vectors expressing human sodium/iodide symporter (hNIS) or luciferase (Luc) under the control of cytomegalovirus (CMV) promoter. After treating transfected and control ARO cells with doxorubicin, iodide uptake assay and luciferase assay were performed. Reversed-phase polymerase chain reaction analysis for hNIS and Western blot analysis for IkappaB protein were executed. Electrophoretic mobility-shift assay (EMSA) was performed to evaluate nuclear factor-kappaB (NF-kappaB) binding activity induced by doxorubicin. Scintigraphic and bioluminescent images were acquired and quantitated before and after doxorubicin in a tumor-bearing mouse model. Radioiodide uptake in ARO cells transfected with the NIS gene under the CMV promoter was remarkably enhanced by doxorubicin, and this corresponded to a significant increase in NIS messenger RNA. In addition, luciferase gene upregulation by doxorubicin was also observed in luciferase gene transfected ARO cells. These results were verified by in vivo imaging in a tumor-bearing mouse model. Moreover, transgene expressional enhancement by doxorubicin was observed after transfecting ARO cells with adenoviral vector or plasmid vector, when transgenes were under the control of a CMV promoter. In addition, NF-kappaB, activated by doxorubicin, induced transgene transcription under the control of the CMV promoter, which possesses an NF-kappaB binding site. These findings indicate that doxorubicin enhances transgene expression under the control of the CMV promoter and that doxorubicin might be used as an adjuvant to radioiodine therapy by NIS gene transfer in anaplastic thyroid carcinoma.

Potential Role of Tubulin Acetylation and Microtubule‐Based Protein Trafficking in Familial Dysautonomia

Familial dysautonomia (FD), a disease of the autonomic and sensory nervous systems, involves mutations in the protein IkappaB kinase complex-associated protein, which is a component of the human Elongator acetylase complex. We suggest a hypothesis in which defects in tubulin acetylation and impairment of microtubule-based protein trafficking may be an underlying cause of FD. In addition, an Arabidopsis homolog of the Elongator subunit ELP3 has been found to bind to the alphabeta-tubulin heterodimer, suggesting that alpha-tubulin may be a cytoplasmic target of Elongator acetylase activity. Studies of synergistic double mutants in yeast indicate a novel role for Elongator in cytoskeletal dynamics, although this is probably because of an effect on actin rather than microtubules. Finally, we suggest that tubulin deacetylase inhibitors may prove useful in the treatment of FD.

Dynamics of IkBa Probed by NMR

Dynamics of IkBa Probed by NMR

Effect of a Cyclooxygenase‐2 Inhibitor on Interleukin‐1β–Stimulated Activation of the Transcription Factor Nuclear Factor‐Kappa B in Human Gingival Fibroblasts

In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1beta-stimulated prostaglandin E(2) (PGE(2)) production almost completely while partially inhibiting IL-6 production in aggressive periodontitis (AgP) human gingival fibroblasts. PGE(2) and the transcription factor nuclear factor-kappa B (NF-kappaB) regulate IL-1beta-stimulated IL-6 production. Cytoplasmic NF-kappaB is bound to inhibitors (IkappaB proteins). IL-1beta initiates a cascade resulting in phosphorylation and degradation of IkappaB, allowing nuclear translocation of NF-kappaB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of IkappaB and NF-kappaB activation. AgP fibroblasts (1 to 2 x 10(6)) were exposed to IL-1beta (1 x 10(11)M) with or without NS-398 (10 nM) in serum-free medium. The NF-kappaB subunit p65 and phospho-IkappaBalpha were measured in whole cell, cytoplasmic, or nuclear extracts, using colorimetric assays. Enzyme-linked immunosorbent assays were used to measure PGE(2) and IL-6 production by 2.5 x 10(4) cells after exposure to IL-1beta with or without NS-398 in serum-free medium. Consistent with previous results, NS-398 reduced IL-1beta-stimulated PGE(2) by approximately 98% (P <0.001) and IL-6 by approximately 65% (P <0.001). IL-1beta increased nuclear and cytoplasmic p65 ( approximately 8-fold [P <0.001] and approximately 2.5-fold [P <0.03], respectively) over control levels. NS-398 reduced IL-1beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1beta increased phospho-IkappaBalpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P <or=0.008). NS-398 inhibited NF-kappaB activation and nuclear p65 levels in human gingival fibroblasts. This seemed to be due to inhibition of the phosphorylation cascade resulting in formation of phospho-IkappaBalpha and free p65. NF-kappaB inhibition may be useful in treating inflammatory diseases such as AgP.

Inhibition of NF-κB Acetylation and its Transcriptional Activity by Daxx

We propose a biochemical mechanism by which Daxx modulates NF-κB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-κB in HeLa cells. Overexpression of Daxx repressed the expression of NF-κB-regulated genes such as Iκ Bα and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-κB stimulated by TNFα. Here, we suggest that Daxx-mediated repression of NF-κB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-κB transcriptional activity.

TNF‐related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle‐wasting cytokine

TWEAK cytokine has been implicated in several biological responses including inflammation, angiogenesis, and osteoclastogenesis. We have investigated the role of TWEAK in regulating skeletal muscle mass. Addition of soluble TWEAK protein to cultured myotubes reduced the mean myotube diameter and enhanced the degradation of specific muscle proteins such as CK and MyHCf. The effect of TWEAK on degradation of MyHCf was stronger than its structural homologue, TNF-alpha. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin-1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphorylation of Akt kinase and its downstream targets GSK-3beta, FOXO1, mTOR, and p70S6K. Furthermore, TWEAK increased the activation of NF-kappaB transcription factor in myotubes. Adenoviral-mediated overexpression of IkappaB alpha deltaN (a degradation-resistant mutant of NF-kappaB inhibitory protein IkappaB alpha) in myotubes blocked the TWEAK-induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin-proteasome system and NF-kappaB. Finally, muscle-specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin-proteasome and NF-kappaB systems.

A Fourth IκB Protein within the NF-κB Signaling Module

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -epsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.

A homeostatic model of IκB metabolism to control constitutive NF‐κB activity

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-κB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IκB, until an external stimulus triggers IκB degradation through an IκB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-κB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-κB-bound and -unbound IκB proteins that are critical for accurate computational predictions of steady-state IκB protein levels and basal NF-κB activity. Simulations reveal a homeostatic NF-κB signaling module in which differential degradation rates of free and bound pools of IκB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Role of NF-.KAPPA.B in Constitutive Expression of MAIL in Epidermal Keratinocytes

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor kappaBzeta (IkappaBzeta). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor kappaB (NF-kappaB)-Bay11-7082 and the IkappaBalphaM supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-kappaB components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-kappaB and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.

Two novel transcripts encoding two Ankyrin repeat containing proteins have preponderant expression during the mouse spermatogenesis

The clone 4921537P18 expressed preponderantly in mouse testis was identified by screening the Riken cDNA database, and two new full-length isoforms of this clone, which were named gsarp1 (Gonad Specific Ankyrin Repeat (ANK) Protein 1) and gsarp2, were found and isolated from mouse testis in the course of the research. Both of the GSARP1 and GSARP2 contain an ANK region circular composed by seven ANKs, and their structural feature is very similar to that of the IkappaB family proteins, while IkappaB proteins associate with the transcription factor NF-kappaB via their ANKs in the NF-kappaB pathway. We investigated the expression pattern at the mRNA level by Reverse transcription PCR. The gsarp1 has high expression level in mouse testis, while has low expression level in the ovary, and the gsarp2 is only expressed in mouse testis. The gsarp1 and gsarp2 begin to be detected at the early and later pachytene stage of meiosis separately, while both have high-expression level at the stage of MI and MII. The result of in situ hybridization reveals that the gsarp1 is primarily expressed in spermatocytes, while gsarp2 is expressed in spermatocytes and spermatids. In view of the structural feature and expression pattern of the GSARP1 and GSARP2, we speculate that they may play a certain role in a signal pathway of meiosis.

Effect of anabolic agents on calpastatin promoters in porcine skeletal muscle and their responsiveness to cyclic adenosine monophosphate-and calcium-related stimuli1

The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5'region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the beta-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium (Ca2+)-related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, IkappaB alpha, and NFkappaB by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and Ca2+-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters.

Unravelling the complexities of the NF-κB signalling pathway using mouse knockout and transgenic models

The nuclear factor-kappaB (NF-kappaB) signalling pathway serves a crucial role in regulating the transcriptional responses of physiological processes that include cell division, cell survival, differentiation, immunity and inflammation. Here we outline studies using mouse models in which the core components of the NF-kappaB pathway, namely the IkappaB kinase subunits (IKKalpha, IKKbeta and NEMO), the IkappaB proteins (IkappaBalpha, IkappaBbeta, IkappaBvarepsilon and Bcl-3) and the five NF-kappaB transcription factors (NF-kappaB1, NF-kappaB2, c-Rel, RelA and RelB), have been genetically manipulated using transgenic and knockout technology.