Regulation of adipose maturation and energy homeostasis.

The adipose tissue of mammals represents a dynamic organ disseminated throughout the body. It fluctuates in abundance according to the availability of metabolic energy supplies. Mature adipose tissue communicates with the central nervous system via a hormonal circuit that controls satiety. Adipogenesis can be recapitulated in cell culture, thus facilitating molecular biological studies of the regulatory proteins that control this process. Such studies have led to the identification of two families of transcription factors that regulate adipogenesis and mammalian energy homeostasis.

Protein kinase B regulates T lymphocyte survival, nuclear factor kappaB activation, and Bcl-X(L) levels in vivo.

The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. We have generated transgenic mice expressing a constitutively active form of PKB (gag-PKB) to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB displayed increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolonged the survival of CD4(+)CD8(+) double positive (DP) thymocytes in fetal thymic organ culture, but was unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I-restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen-specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB displayed elevated levels of the antiapoptotic molecule Bcl-X(L). In addition, the activation of peripheral T cells led to enhanced nuclear factor (NF)-kappaB activation via accelerated degradation of the NF-kappaB inhibitory protein IkappaBalpha. Our data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules (PKB, Bcl-X(L), and NF-kappaB) in vivo in T lymphocytes.

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand.

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.

Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.

Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies.

Impaired TNFalpha-induced A20 expression in E1A/Ras-transformed cells.

BACKGROUND: Tumour necrosis factor (TNF) is capable of activating the cell death pathway, and has been implicated in killing transformed cells. However, TNF also activates survival signals, including NF-kappaB activation and the subsequent expression of anti-apoptotic genes, leading to protection against TNF toxicity. METHODS: In this study, we show that, although untransformed mouse embryonic fibroblasts (MEFs) were resistant to TNF killing, E1A/Ras-transformed MEFs were susceptible to extensive apoptosis induced by TNF. The key factors for determining TNF sensitivity were explored by comparing wild-type and E1A/Ras-transformed MEFs. RESULTS: TNF signalling to NF-kappaB and to its target genes such as IkappaBalpha seemed to be mostly intact in E1A/Ras-transformed cells. Instead, the induction of A20 was completely abolished in E1A/Ras-transformed MEFs, although A20 is known to be NF-kappaB dependent. Reintroduction of A20 into E1A/Ras-transformed MEFs rescued these cells from TNF-induced death and reduced the formation of the FADD/caspase-8 complex. This impaired A20 induction in E1A/Ras MEFs was not because of the stabilisation of p53 or a defective TNF-induced p38 and Jun N-terminal kinase (JNK) signalling. Consistently, we found a reduced A20 promoter activity but normal NF-kappaB activity in TNF-treated E1A/Ras MEFs. However, Bcl-3 seemed to have a role in the transactivation of the A20 promoter in E1A/Ras cells. CONCLUSIONS: Our results suggest that specific inhibition of certain survival factors, such as A20, may determine the sensitivity to TNF-induced apoptosis in transformed cells such as E1A/Ras MEFs.

FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis.

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.

Mechanism of ASC-mediated apoptosis: bid-dependent apoptosis in type II cells.

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor molecule that mediates apoptotic and inflammatory signals, and implicated in tumor suppression. However, the mechanism of ASC-mediated apoptosis has not been well elucidated. Here, we investigated the molecular mechanisms of ASC-mediated apoptosis in several cell lines using a caspase recruitment domain 12-Nod2 chimeric protein that transduces the signal from muramyl dipeptide into ASC-mediated apoptosis. Experiments using dominant-negative mutants, small-interfering RNAs and peptide inhibitors for caspases indicated that caspase-8 was generally required for ASC-mediated apoptosis, whereas a requirement for caspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like inhibitor protein, a natural caspase-8 inhibitor, suppressed ASC-mediated apoptosis, and Clarp-/- mouse embryonic fibroblasts were highly sensitive to ASC-mediated apoptosis. Bax-deficient HCT116 cells were resistant to ASC-mediated apoptosis as reported previously, although we failed to observe colocalization of ASC and Bax in cells. Like Fas-ligand-induced apoptosis, the ASC-mediated apoptosis was inhibited by Bcl-2 and/or Bcl-XL in type-II but not type-I cell lines. Bid was cleaved upon ASC activation, and suppression of endogenous Bid expression using small-interfering RNAs in type-II cells reduced the ASC-mediated apoptosis. These results indicate that ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8.

Mediation of TNF receptor-associated factor effector functions by apoptosis signal-regulating kinase-1 (ASK1).

Tumor necrosis factor-alpha (TNF), a major inflammatory cytokine, generates a wide variety of cellular responses via key cytoplasmic adaptor molecules named TNF receptor-associated factors (TRAFs). We report that TRAF2, TRAF5 and TRAF6 associate with apoptosis signal-regulating kinase 1 (ASK1), and a catalytically-inactive ASK1 mutant blocks stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) activation by these TRAFs. A truncated derivative of TRAF2, which inhibits SAPK activation by TNF, blocks TNF-induced ASK1 activation. Furthermore, protection from TNF-induced cell death conferred by an ASK1 mutant is dependent upon TRAF2. Hence, ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling, and the TRAF2 - ASK1 connection completes the signaling cascade from TNF to SAPK/JNK activation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS.

Tumor necrosis factor receptor-associated factors (TRAFs) are adaptor proteins important in mediating intracellular signaling. We report here that targeted deletion of traf6 greatly increases the frequency of failure of neural tube closure and exencephaly in traf6 (-/-) mice. The penetrance of this defect is influenced by genetic background. Neural tube fusion requires the coordination of several biological processes, including cell migration invoked by contact-dependent signaling, cell proliferation, and programmed cell death (PCD). To gain greater insight into the role of TRAF6 in these processes, neural development and migration within the CNS of traf6 (-/-) mice and controls were assessed through temporal examination of a number of immunohistochemical markers. In addition, relative levels of cellular proliferation and PCD were examined throughout embryonic development using bromodeoxyuridine (BrdU) and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotinylated nick end labeling (TUNEL), respectively. The data suggest that loss of TRAF6 does not significantly alter the level of cellular proliferation or the pattern of neural differentiation per se, but rather regulates the level of PCD within specific regions of the developing CNS. Substantial reductions in TUNEL were observed within the ventral diencephalon and mesencephalon in exencephalic traf6 (-/-) embryos. Our results demonstrate a novel and prominent role for TRAF6 in the regional control of PCD within the developing CNS.

Murine TMJ loading causes increased proliferation and chondrocyte maturation.

The purpose of this study was to examine the effects of forced mouth opening on murine mandibular condylar head remodeling. We hypothesized that forced mouth opening would cause an anabolic response in the mandibular condylar cartilage. Six-week-old female C57BL/6 mice were divided into 3 groups: (1) control, (2) 0.25 N, and (3) 0.50 N of forced mouth opening. Gene expression, micro-CT, and proliferation were analyzed. 0.5 N of forced mouth opening caused a significant increase in mRNA expression of Pthrp, Sox9, and Collagen2a1, a significant increase in proliferation, and a significant increase in trabecular spacing in the subchondral bone, whereas 0.25 N of forced mouth opening did not cause any significant changes in any of the parameters examined. Forced mouth opening causes an increase in the expression of chondrocyte maturation markers and an increase in subchondral trabecular spacing.

M(1)-like muscarinic acetylcholine receptors regulate fast-spiking interneuron excitability in rat dentate gyrus.

Cholinergic transmission through muscarinic acetylcholine receptors (mAChRs) plays a key role in cortical oscillations. Although fast-spiking (FS), parvalbumin-expressing basket cells (BCs) are proposed to be the cellular substrates of gamma oscillations, previous studies reported that FS nonpyramidal cells in neocortical areas are unresponsive to cholinergic modulation. Dentate gyrus (DG) is an independent gamma oscillator in the hippocampal formation. However, in contrast to other cortical regions, the direct impact of mAChR activation on FS BC excitability in this area has not been investigated. Here, we show that bath-applied muscarine or carbachol, two mAChR agonists, depolarize DG BCs in the acute brain slices, leading to action potential firing in the theta-gamma bands in the presence of blockers of ionotropic glutamate and gamma-aminobutyric acid type A receptors at physiological temperatures. The depolarizing action persists in the presence of tetrodotoxin, a voltage-gated Na(+) channel blocker. In voltage-clamp recordings, muscarine markedly reduces background K(+) currents. These effects are mimicked by oxotremorine methiodide, an mAChR-specific agonist, and largely reversed by atropine, a non-selective mAChR antagonist, or pirenzepine, an M(1) receptor antagonist, but not by gallamine, an M(2/4) receptor antagonist. Interestingly, in contrast to M(1)-receptor-mediated depolarization, M(2) receptor activation by the specific agonist arecaidine but-2-ynyl ester tosylate down-regulates GABA release at BC axons-the effect is occluded by gallamine, an M(2) receptor antagonist. Overall, muscarinic activation results in a net increase in phasic inhibitory output to the target cells. Thus, cholinergic activation through M(1)-like receptor enhances BC activity and promotes the generation of nested theta and gamma rhythms, thereby enhancing hippocampal function and associated performance.

Analysis of microarchitectural changes in a mouse temporomandibular joint osteoarthritis model.

OBJECTIVE: Little is known about the natural progression of the disease process of temporomandibular joint (TMJ) osteoarthritis (OA), which affects approximately 1% of the US population. The goal of this study was to examine the early microarchitectural and molecular changes in the condylar cartilage and subchondral bone in biglycan/fibromodulin (Bgn/Fmod) double-deficient mice, which develop TMJ-OA at 6 months. METHODS: TMJs from 3-month-old (n=44) and 9-month-old (n=52) wild-type (WT n=46) and Bgn/Fmod (n=50) double-deficient mice were evaluated. Micro-CT analysis of the subchondral bone (n=24), transmission electron microscopy for condylar cartilage fibril diameters (n=26), and real-time PCR analysis for gene expression for bone and cartilage maturation markers (n=45) was performed. RESULTS: A statistically significant increase in collagen fibril diameter of the condylar cartilage and a decrease in expression of Parathyroid related protein in the mandibular condylar head were observed in the 3-month Bgn/Fmod double-deficient mice compared to WT controls. The 9-month Bgn/Fmod double-deficient mouse demonstrated an increase in bone volume and total volume in subchondral bone, and an increase in the expression of Collagen Type X and Aggrecan in the mandibular condylar head compared to the WT controls. CONCLUSION: We found that changes in the microarchitecture of the condylar cartilage preceded changes in the subchondral bone during OA in the TMJ in Bgn/Fmod double-deficient mice.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.

Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBP delta. Likewise, MIX is a direct inducer of C/EBP beta gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBP delta and C/EBP beta accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP alpha. Conclusive evidence has already shown that C/EBP alpha regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBP delta and C/EBP beta play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP alpha. Conditions facilitating the precocious expression of either C/EBP delta or C/EBP beta were observed to accelerate adipogenesis and, in the case of C/EBP beta, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP beta effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBP beta in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat-laden adipocytes.

Gene targeting in the analysis of mammalian apoptosis and TNF receptor superfamily signaling.

Apoptosis, or programmed cell death (PCD), is the subject of much current investigative interest. Developing embryos and many adult organ systems require the tight coupling of cellular proliferation and PCD to ensure proper organogenesis and optimal tissue function. Over the past decade, our knowledge of the genetic basis underlying the execution of apoptosis in mammals has progressed enormously, thanks largely to groundbreaking studies performed in the nematode Caenorhabditis elegans. In contrast, the components of the signaling apparatus that links the various death stimuli and the receptors they stimulate to the execution mechanism remain relatively unknown. It is only in the past 4 years that studies of signal transduction via members of the tumor necrosis factor (TNF) receptor superfamily have identified a plethora of novel signaling proteins, including molecules that are directly involved in apoptosis signaling, and others that regulate the induction of cell death. This two-part review focuses on the biology of apoptosis and signaling through members of the TNF receptor superfamily as revealed by the study of gene-targeted "knockout" mice. These genetic mutant animals are invaluable tools not only for confirming or refuting a proposed function of a particular gene in an in vivo setting, but also for uncovering novel functions for a gene that were not anticipated from conventional in vitro experiments. In the field of apoptosis, as for many other areas of biomedical research, knockout mice and cell lines can be used as models for studying human disease, with the ultimate goal of developing therapeutic strategies.

Requirement of FADD for tumor necrosis factor-induced activation of acid sphingomyelinase.

The generation of mice strains deficient for select members of the signaling complex of the 55-kDa tumor necrosis factor receptor (TNF-R55) has allowed the assignment of specific cellular responses to distinct TNF-R55-associated proteins. In particular, the TNF-R55-associated protein FADD seems to be responsible for recruitment and subsequent activation of caspase 8. In this report we demonstrate the requirement of FADD for TNF-induced activation of endosomal acid sphingomyelinase (A-SMase). In primary embryonic fibroblasts from FADD-deficient mice the activation of A-SMase by TNF-R55 ligation was almost completely impaired. This effect is specific in that other TNF responses like activation of NF-kappaB or neutral (N-)SMase remained unaffected. In addition, interleukin-1-induced activation of A-SMase in FADD-deficient cells was unaltered. In FADD-/- embryonic fibroblasts reconstituted by transfection with a FADD cDNA expression construct, the TNF responsiveness of A-SMase was restored. The results of this study suggest that FADD, in addition to its role in triggering a proapoptotic caspase cascade, is required for TNF-induced activation of A-SMase.

Identification and characterization of an immunophilin expressed during the clonal expansion phase of adipocyte differentiation.

Mouse 3T3-L1 cells differentiate into fat-laden adipocytes in response to a cocktail of adipogenic hormones. This conversion process occurs in two discrete steps. During an early clonal expansion phase, confluent 3T3-L1 cells proliferate and express the products of the beta and delta members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. The cells subsequently arrest mitotic growth, induce the expression of the alpha form of C/EBP, and acquire the morphology of fully differentiated adipocytes. Many of the genes induced during the terminal phase of adipocyte conversion are directly activated by C/EBP alpha, and gratuitous expression of this transcription factor is capable of catalyzing adipose conversion in a number of different cultured cell lines. The genetic program undertaken during the clonal expansion phase of 3T3-L1 differentiation, controlled in part by C/EBP beta and C/EBP delta, is less clearly understood. To study the molecular events occurring during clonal expansion, we have identified mRNAs that selectively accumulate during this phase of adipocyte conversion. One such mRNA encodes an immunophilin hereby designated FKBP51. In this report we provide the initial molecular characterization of FKBP51.

FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4.

Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.

Low frequency of bacteremia after endoscopic mucosal resection.

BACKGROUND: Endoscopic mucosal resection has become a popular alternative for the treatment of early-stage neoplasia of the gastrointestinal tract. However, there are still no data on the frequency of bacteremia associated with this form of treatment. METHODS: We conducted a prospective study of 21 men and 17 women undergoing endoscopic mucosal resection with a cap-fitted panendoscope for upper gastrointestinal lesions. Blood cultures were performed before, 10 minutes after, and 4 hours after the procedure for both aerobic and anaerobic bacteria. RESULTS: Blood culture at baseline was negative in all the patients. Two of 38 patients (5.3 %) had positive blood culture at 10 minutes after the procedure. The isolated microorganisms were Streptococcus salivarius and Corynebacterium species. All patients had negative blood cultures 4 hours later. None of these 38 patients had any symptoms or signs associated with infection. CONCLUSIONS: Bacteremia associated with endoscopic mucosal resection is infrequent and transient.