Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.     

Constitutive nuclear expression of the I kappa B kinase complex and its activation in human neutrophils

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus.     

Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.     

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase

STO-609, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase kinase (CaM-KK) was synthesized, and its inhibitory properties were investigated both in vitro and in vivo. STO-609 inhibits the activities of recombinant CaM-KK alpha and CaM-KK beta isoforms, with K(i) values of 80 and 15 ng/ml, respectively, and also inhibits their autophosphorylation activities. Comparison of the inhibitory potency of the compound against various protein kinases revealed that STO-609 is highly selective for CaM-KK without any significant effect on the downstream CaM kinases (CaM-KI and -IV), and the IC(50) value of the compound against CaM-KII is approximately 10 microg/ml. STO-609 inhibits constitutively active CaM-KK alpha (glutathione S-transferase (GST)-CaM-KK-(84-434)) as well as the wild-type enzyme. Kinetic analysis indicates that the compound is a competitive inhibitor of ATP. In transfected HeLa cells, STO-609 suppresses the Ca(2+)-induced activation of CaM-KIV in a dose-dependent manner. In agreement with this observation, the inhibitor significantly reduces the endogenous activity of CaM-KK in SH-SY5Y neuroblastoma cells at a concentration of 1 microg/ml (approximately 80% inhibitory rate). Taken together, these results indicate that STO-609 is a selective and cell-permeable inhibitor of CaM-KK and that it may be a useful tool for evaluating the physiological significance of the CaM-KK-mediated pathway in vivo as well as in vitro.     

Overexpression of RelA in transgenic mouse thymocytes: specific increase in levels of the inhibitor protein I kappa B alpha.

RelA (p65) is one of the strongest activators of the Rel/NF-kappa B family. As a first step to elucidate the mechanisms that regulate its activity in vivo, we have generated transgenic mice overexpressing RelA in the thymus. Although the levels of RelA were significantly increased in thymocytes of transgenic mice, the overall NF-kappa B-binding activity in unstimulated cells was not augmented compared with that in control thymocytes. This could be explained by the dramatic increase of endogenous I kappa B alpha levels observed in RelA-overexpressing cells in both cytoplasmic and nuclear compartments. The ikba mRNA levels were not augmented by overexpressed RelA, but I kappa B alpha inhibitor was found to be stabilized through association with RelA. Although a fraction of RelA was associated with cytoplasmic p105, no changes in the precursor levels were observed. Upon stimulation of RelA-overexpressing thymocytes with phorbol 12-myristate 13-acetate and lectin (phytohemaglutinin), different kappa B-binding complexes, including RelA homodimers, were partially released from I kappa B alpha. Association of RelA with I kappa B alpha prevented complete degradation of the inhibitor. No effect of phorbol 12-myristate 13-acetate-lectin treatment was detected on RelA associated with p105. Our data indicate that cytoplasmic retention of overexpressed RelA by I kappa B alpha is the major in vivo mechanism controlling the potential excess of NF-kappa B activity in long-term RelA-overexpressing thymocytes.     

Cyclin-dependent kinase inhibitor Cdkn2c regulates B cell homeostasis and function in the NZM2410-derived murine lupus susceptibility locus Sle2c1

Sle2c1 is an NZM2410- and NZB-derived lupus susceptibility locus that induces an expansion of the B1a cell compartment. B1a cells have a repertoire enriched for autoreactivity, and an expansion of this B cell subset occurs in several mouse models of lupus. A combination of genetic mapping and candidate gene analysis presents Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18(INK4c) (p18), as the top candidate gene for inducing the Slec2c1-associated expansion of B1a cells. A novel single nucleotide polymorphism in the NZB allele of the Cdkn2c promoter is associated with a significantly reduced Cdkn2c expression in the splenic B cells and peritoneal cavity B1a cells from Sle2c1-carrying mice, which leads to a defective G1 cell cycle arrest in splenic B cells and increased proliferation of peritoneal cavity B1a cells. As the cell cycle is differentially regulated in B1a and B2 cells, these results suggest that Cdkn2c plays a critical role in B1a cell self-renewal and that its impaired expression leads to an accumulation of these cells with high autoreactive potential.     

CD40 signaling in B cells regulates the expression of the Pim-1 kinase via the NF-kappa B pathway.

The ability of CD40 signaling to regulate B cell growth, survival, differentiation, and Ig class switching involves many changes in gene expression. Using cDNA expression arrays and Northern blotting, we found that CD40 signaling increased the mRNA levels for pim-1, a protooncogene that encodes a serine/threonine protein kinase. Subsequent experiments showed that CD40 engagement also increased both Pim-1 protein levels and Pim-1 kinase activity in B cells. We then investigated the signaling pathways by which CD40 regulates Pim-1 expression and found that CD40 up-regulates Pim-1 primarily via the activation of NF-kappaB. Inhibiting the activation of NF-kappaB, either by treating cells with a chemical inhibitor, BAY11-7082, or by inducibly expressing a superrepressor form of IkappaBalpha, significantly impaired the ability of CD40 to increase Pim-1 protein levels. Because Pim-1 expression is associated with cell proliferation and survival, we asked whether this correlated with the ability of CD40 signaling to prevent anti-IgM-induced growth arrest in the WEHI-231 murine B cell line, a model for Ag-induced clonal deletion. We found that the anti-IgM-induced growth arrest in WEHI-231 cells correlated with a substantial decrease in Pim-1 levels. In contrast, culturing WEHI-231 cells with either anti-CD40 Abs or with the B cell mitogen LPS, both of which prevent the anti-IgM-induced growth arrest, also prevented the rapid decline in Pim-1 levels. This suggests that Pim-1 could regulate the survival and proliferation of B cells.     

不同剂量瑞舒伐他汀对ACI患者外周血淋巴细胞OX40L表达及血清OX40L和hs-CRP水平的影响

目的:探讨不同剂量瑞舒伐他汀对急性动脉硬化脑梗死(ACI)病人外周血淋巴细胞OX40L及血清OX40L和hs-CRP水平的影响.方法:选择40名动脉硬化性脑梗死病人,随机分为瑞舒伐他汀20 mg治疗组(A组)和瑞舒伐他汀10 mg治疗组(B组),应用QRT-PCR及酶联免疫吸附试验(ELISA)法分别检测外周血淋巴细胞OX40L及血清OX40L和hs-CRP的水平.结果:A组与B组外周血淋巴细胞OX40L下降幅度比较(P＜0.01)及血清sOX40L和CRP水平治疗后有明显降低(P＜0.05),A组与B组比较A组降低更加明显,有统计学意义.结论:动脉硬化性脑梗死病人急性期服用瑞舒伐他汀20 mg能更明显的降低患者外周血淋巴细胞OX40L及血清OX40L和CRP的水平,提示动脉粥样硬化性脑梗死病人早期服用较大剂量的他汀药物治疗效果可能更好.     

Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)₃AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.     

The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.     

Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa.

The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.     

Association of cyclin-dependent kinase inhibitor 2A/B with increased risk of developing breast cancer

There is a growing body of data reporting the association of genetic alterations in chromosome 9P21 with the risk of developing cancer. In the current study, we studied the association of a genetic variant in CDKN2A/B, rs1333049, with the risk of developing breast cancer. A total of 339 participants with and without breast cancer entered to the study. Genotyping was done by the TaqMan real-time polymerase chain reaction (RT-PCR) method and gene expression analysis was ran by RT-PCR. Our data showed that the minor allele homozygote in the total population was 10%, whereas for heterozygote was 38%. The dominant genetic model demonstrated that individuals with breast cancer had advanced TNM classification. Moreover, the logistic regression revealed that individuals who had CC/CG genotypes might have an enhanced risk of developing breast cancer when compared to the holders of GG genotype (e.g., OR = 2.8; 95% CI,1.4–5.4; p = .001), after regulated for confounders; age and body mass index. Furthermore, our analysis showed that the CDKN2A/B gene was downregulated in patients (p < .001). We showed a meaningful relationship of CDKN2A/B with the risk of breast cancer, cancer, showing the importance of studies in great sample size and several centers for studying the value of the marker as a risk classification in the management of patients with breast cancer.     

Basophil-associated OX40 Ligand Participates in the Initiation of Th2 Responses during Airway Inflammation

Asthma is characterized by increased airway submucosal infiltration of T helper (Th) cells and myeloid cells that co-conspire to sustain a chronic inflammation. While recent studies have demonstrated that the myeloid basophils promote Th2 cells in response to various types of allergens, the underlying mechanisms are poorly understood. Here, we found for the first time that in a mouse model of allergic asthma basophils highly expressed OX40 ligand (OX40L) after activation. Interestingly, blockade of OX40-OX40L interaction suppressed basophils-primed Th2 cell differentiation in vitro and ameliorated ovalbumin (OVA)-induced allergic eosinophilic inflammation mediated by Th2 activation. In accordance, the adoptive transfer of basophils derived from mediastinal lymph nodes (MLN) of OVA-immunized mice triggered a robust Th2 response and eosinophilic inflammation in wild-type mice but largely muted in OX40^−/− mice and mice receiving OX40L-blocked basophils. Taken together, our results reveal a critical role of OX40L presented by the activated basophils to initiate Th2 responses in an allergic asthma model, implicating OX40-OX40L signaling as a potential therapeutic target in the treatment of allergic airway inflammation.     

Highly B lymphocyte‐specific tamoxifen inducible transgene expression of CreERT2 by using the LC‐1 locus BAC vector

The generation of cell type specific inducible Cre transgenic mice is the most challenging and limiting part in the development of spatio‐temporally controlled knockout mouse models. Here we report the generation and characterization of a B lymphocyte‐specific tamoxifen‐inducible Cre transgenic mouse strain, LC‐1‐hCD19‐CreER^T2. We utilized the human CD19 promoter for expression of the tamoxifen‐inducible Cre recombinase (CreER^T2) gene, embedded in genomic sequences previously reported to give minimal position effects after transgenesis. Cre recombinase activity was evaluated by cross‐breeding the LC‐1‐hCD19‐CreER^T2 strain with a strain containing a floxed gene widely expressed in the hematopoietic system. Cre activity was only detected in the presence of tamoxifen and was restricted to B lymphocytes. The efficacy of recombination ranged from 27 to 61% in the hemizygous and homozygous mice, respectively. In conclusion, the LC‐1‐hCD19‐CreER^T2 strain is a powerful tool to study gene function specifically in B lymphocytes at any chosen time point in the lifecycle of the mouse. genesis 47:729–735, 2009. © 2009 Wiley‐Liss, Inc.