Heat-labile Factor Research Articles

Evidence for a major role of endogenous fibroblast growth factor‐2 in apoptotic cortex‐induced subventricular zone cell proliferation

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) of lateral ventricles. It is well established that cortical damage leads to SVZ cell proliferation and neuronal differentiation. We have previously demonstrated in rat that, when treated with the apoptosis-inducing agent staurosporine, cortex explants release heat-labile factors that promote SVZ cell culture proliferation. In the present report, we investigated in vitro mechanisms involved in cortex injury-triggered neurogenesis in the rat. We demonstrated, using immunoblotting analysis and fibroblast growth factor (FGF)-2 enzyme-linked sandwich immunosorbent assay, that treatment of cortex explants with apoptosis-inducing agents increases the release of FGF-2. We next determined the effects of apoptotic cortex-released factors in regulating SVZ cell proliferation and neuronal differentiation by using bromodeoxyuridine incorporation and microtubule-associated protein 2 immunostaining assays, respectively. We found that conditioned media derived from staurosporine-treated cortex explants enhanced SVZ cell culture proliferation and differentiation by over 50 and 80%, respectively. Finally, we showed that immunodepletion of FGF-2 or pharmacological blockade of FGF-2 receptor by SU5402 completely abolished staurosporine-treated cortex mitogenic activity on SVZ cultures but did not alter its activity on neuronal cell differentiation. Altogether, the present report establishes that the release of endogenous FGF-2 by apoptotic cortex explants plays a major role in the induction of SVZ cell proliferation but not neuronal differentiation, which probably depends on the release of other as yet unidentified cortical factors.

Role of gonadotropin and Leydig cell-secreted factors in the control of testicular macrophage activities in the wall lizard Hemidactylus flaviviridis

The present in vitro study for the first time demonstrates the endocrine and paracrine control of testicular macrophage activities in ectodermic vertebrates. Follicle-stimulating hormone (FSH) increased phagocytosis and superoxide production by macrophages. In regard to paracrine control, non-activated Leydig cell-conditioned medium (LCCM) decreased both the activities, whereas FSH-preactivated LCCM had differential effects: inhibitory on phagocytosis and stimulatory on superoxide production. However, FSH-activated LCCM, in addition to superoxide production, also enhanced phagocytosis. After heat inactivation, FSH-activated LCCM inhibited both the activities. Addition of FSH resulted in stimulation of phagocytosis, while partially restored the superoxide production. It can be speculated that androgen in heat-inactivated FSH-activated LCCM, in the presence of FSH, instead of inhibitory had stimulatory effect on phagocytosis, but remained inhibitory to superoxide production. Further, FSH-induced Leydig cell-secreted non-steroidal heat-labile factors appear to have stimulatory effect on superoxide production. This was corroborated by experiments with dihydrotestosterone in presence/absence of FSH.

Role of heat-labile serum factor or host complement in the inhibition ofPlasmodium falciparumsporogonic stages inAnopheles stephensiby gametocyte carriers' serological factors

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Formation of the 3′ end of histone mRNA: Getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3′ end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem–loop structure. This distinct 3′ end is generated by specialized 3′ end processing machinery that cleaves histone pre-mRNAs 4–5 nucleotides downstream of the stem–loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3′ end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.

Induction of a Soluble Anti-HIV-1 factor (s) with IFN-γ, IL-10, and β-Chemokine Modulating Activity by an Influenza-Bacterial Polyantigenic Mixture

Partial immune restoration may be obtained with highly active antiretroviral therapy (HAART), but specific anti-HIV-1 immune responses do not appear to improve substantially. We have demonstrated that a soluble factor(s) induced by a mixture of inactivated influenza and bacterial vaccines called polyantigenic immunomodulator (PAI), possesses strong immunoregulatory and anti-HIV-1 activities. In the present study, we show that culture fluids from both PAI-stimulated peripheral blood mononuclear cells (PBMC) and CD8+ T-cells of HIV-1 infected patients were able to suppress HIV-1 replication in an MHC-unrestricted fashion. The PAI-induced antiviral activity was eliminated when culture fluids were pre-heated at 100°C for 10 min. and it is associated with induction of IFN-γ, MIP-1α, MIP-1β, and RANTES production, but inhibition of IL-10. Furthermore, this induction is dependent on the immunological status (CD4:CD8 ratio) of the HIV-1 infected patient. Taken together, our results suggest that the MHC-unrestricted HIV-1 suppression that is induced by culture fluids from PAI-stimulated PBMC may result from the stimulation of immune cell subpopulations to produce a heat-labile antiviral soluble factor(s), which in turn modulate cytokine and β-chemokine production. The identification of this PAI-induced soluble factor(s) may have major therapeutic potential.

Bystander immuno-modulatory effects of human neural stem cell line, HB1.F3, on lymphocyte proliferation induced by mitogens and alloantigens (81.9)

Abstract A stable clonal cell line of human neural stem cells (NSCs), HB1.F3, has been shown therapeutic effect on acute stroke or intracerebral haemorrhage by the mechanisms of cell replacement and several bystander effects. To investigate the bystander immunomodulatory functions of HB1.F3 cells, we examined immunosuppressive effects of HB1.F3 on T cell effector functions in vitro. We found that heat-labile soluble factors from hNSCs culture supernatant are attributed to the inhibition of peripheral blood lymphocytes (PBLs) proliferation induced by mixed lymphocyte reaction (MLR) or phorbol myristate acetate (PMA)/ionomycin. In contrast to inhibitory effect of HB1.F3, they promoted cytokine secretion from PBLs including IFN-r, TNF-a, IL-10, IL-6 during mixed lymphocyte reaction by soluble factor dependant-manner. HB1.F3 cell line expressed TGF-beta and Trail mRNA, not FasL. we confirmed that the apoptosis induction of PBLs by HB1.F3 is one of the mechanisms of T cell proliferation inhibition during MLRs. In summary, HB1.F3 cells could suppress the T cell proliferation by induction of apoptosis and promote both inflammatory and immune suppressive cytokines. This study suggested that therapeutically effective hNSCs cell line on several stroke models might be due to the bystander immunomodulatory functions by the secretion of immunosuppressive soluble factors.

The subventricular zone releases factors which can be protective in oxygen/glucose deprivation-induced cortical damage: An organotypic study

A number of studies have already established the role of the subventricular zone in sustaining adult neurogenesis in different brain regions and under different pathological conditions, but nothing is reported about the role of this germinal area in preserving cell viability. In this work, we developed an organotypic culture model of the forebrain structures that comprise the neocortex, striatum, subventricular zone, and corpus callosum. With this model, we investigated the role of the subventricular zone in modulating cell viability in the cortex after oxygen/glucose deprivation. Here we have demonstrated that soluble heat-labile factors released by the subventricular zone in the media can lead to protection specifically in the cortical area. No protection was observed when medium, conditioned with factors released during the insult was administered to the hippocampal slices. Moreover, the use of different modifications of the slice cultures showed that the removal of the subventricular zone increased the cellular damage induced by oxygen/glucose deprivation. Furthermore, by using pharmacological experiments to investigate the possible mechanisms that regulate this subventricular function, we found evidence of purinergic involvement. We postulate that extracellular ATP signaling in the subventricular zone exacerbates cortical damage induced by hypoxia/hypoglycemia. For the first time, we demonstrate in vitro that the germinal subventricular zone can release factors that can be protective after exposure to a metabolic stressor. These released factors are not yet characterized but we identified in the extracellular ATP a factor that may interfere with the protective role of the subventricular zone during metabolic cortical damage.

Amino acid content of soybean samples collected in different Brazilian states: harvest 2003/2004

Soybean is the most important protein source in animal nutrition and is widely used in poultry diets. Several factors influence the concentration of amino acids present in soybean grains cultivated in Brazil, such as climatic changes, genetics, topography, and soil fertility. Many technologies of soybean processing are used in order to eliminate or inactivate both heat-labile and heat-stable anti-nutritional factors. During processing, soybean by-products with different nutritional values are also produced. Processing conditions may as well affect the coefficients of digestibility of nutrients that are present in soybean protein sources. Furthermore, positive and negative effects of the environment may be masked by changes in processing conditions. This study aimed to compare the levels of crude protein and essential and non-essential amino acids in soybean samples collected in the states of Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, and Santa Catarina during the harvest of 2003/2004 in Brazil. Measurements were made using NIRS (Near Infrared System of Reflectance Spectroscopy), and expressed on an "as-fed" basis. Soybeans sampled in different states evidenced nutritional differences. It is worth noting that samples collected in Minas Gerais, Rio Grande do Sul and Mato Grosso do Sul presented high levels of essential amino acids. The analyzed levels of essential amino acids were not always directly related to the protein concentrations of the samples. Because of the diversity of growing conditions in Brazil and worldwide, soybean produced in different environmental conditions is expected to show variable protein composition and quality, as demonstrated in the present study.

Improving the bioavailability of nutrients in plant foods at the household level

Plant foods are the major staples of diets in developing countries, in which the consumption of animal-source foods is often low because of economic and/or religious concerns. However, such plant-based diets are often associated with micronutrient deficits, exacerbated in part by poor micronutrient bioavailability. Diet-related factors in plant foods that affect bioavailability include: the chemical form of the nutrient in food and/or nature of the food matrix; interactions between nutrients and other organic components (e.g. phytate, polyphenols, dietary fibre, oxalic acid, protein, fat, ascorbic acid); pretreatment of food as a result of processing and/or preparation practices. Consequently, household strategies that reduce the content or counteract the inhibiting effects of these factors on micronutrient bioavailability are urgently needed in developing-country settings. Examples of such strategies include: germination, microbial fermentation or soaking to reduce the phytate and polyphenol content of unrefined cereal porridges used for young child feeding; addition of ascorbic acid-containing fruits to enhance non-haem-Fe absorption; heating to destroy heat-labile anti-nutritional factors (e.g. goitrogens, thiaminases) or disrupt carotenoid-protein complexes. Such strategies have been employed in both experimental isotope-absorption and community-based studies. Increases in Fe, Zn and Ca absorption have been reported in adults fed dephytinized cereals compared with cereals containing their native phytate. In community-based studies in rural Malawi improvements in dietary quality and arm-muscle area and reductions in the incidence of anaemia and common infections in young children have been observed.

Modification of a Mammalian Cell Protein in the Presence of [ 32 P-Adenylate]NAD: Evidence for ADP Ribosylation Activity Associated with Helicobacter pylori

Culture filtrates from Helicobacter pylori promote the transfer of the radiolabel from [32P-adenylate]NAD to one or more heat-labile factors within extracts prepared from several mammalian cell lines, with the predominate radiolabeled species exhibiting an apparent molecular mass of greater than 130 kDa. Our results suggest that several H. pylori strains release a factor that ADP-ribosylates a mammalian target protein.

Endogenous factors derived from embryonic cortex regulate proliferation and neuronal differentiation of postnatal subventricular zone cell cultures

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex-derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat-labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex-derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)-2- but not epidermal growth factor (EGF)-immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF-2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.

RNA determinants and protein components of the histone pre‐mRNA processing machinery

Most metazoan mRNAs encoding histones are cleaved but not polyadenylated at their 3′ ends. Cleavage is directed by the U7 small nuclear ribonucleoprotein (U7 snRNP) and requires a heat-labile factor. We recently identified the heat-sensitive complex essential for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor, two subunits of the cleavage stimulation factor and symplekin. To identify which of these factors are essential for cleavage, we are attempting to reconstitute an in vitro processing system with purified and recombinant components. To investigate the role of U7 snRNA in processing, we have determined the RNA requirements for the assembly of a functional U7 snRNP in the Xenopus oocyte system by injecting chimeric histone H4 pre-mRNA / U7 snRNA constructs. We find that the rate limiting step is the assembly of the unique U7 Sm core of proteins, consistent with a role of U7 snRNP in recruiting the additional components of the processing machinery. By Nucleotide Analog Interference Mapping (NAIM) and conventional mutagenesis, we have identified the sequence and structural features of U7 critical for assembly of active particles. These include a flexible 5′-half (revealed by pseudouridine substitution) and four critical nucleotides in the 3′-half of the U7 Sm site, as well as correct placement and length of the U7 hairpin. Research support: HHMI, NIH.

Increased Susceptibility to Apoptosis of CD56dimCD16+ NK Cells Induces the Enrichment of IFN-γ-Producing CD56bright Cells in Tuberculous Pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.

Symplekin and multiple other polyadenylation factors participate in 3′-end maturation of histone mRNAs

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoprotein (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and symplekin. Reconstitution experiments reveal that symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation.

Changes in the haemolymph proteome of Spodoptera littoralis induced by the parasitoid Chelonus inanitus or its polydnavirus and physiological implications

The egg–larval parasitoid Chelonus inanitus induces in its host Spodoptera littoralis two major developmental effects, namely a precocious onset of metamorphosis followed by a developmental arrest in the prepupal stage. Along with each egg, the wasp injects polydnavirus and venom into the host egg. The polydnavirus has been shown to play a major role in inducing the developmental arrest while the parasitoid larva is instrumental in inducing the precocious onset of metamorphosis. Here we report that experimental dilution of haemolymph of polydnavirus-containing larvae can partially prevent the developmental arrest while injection of native, but not of heat-treated, haemolymph or plasma from polydnavirus-containing larvae into nonparasitized larvae could induce developmental arrest in 14–15% of the larvae. This illustrates that heat-labile factors present in haemolymph play a role in causing developmental arrest. Injection of parasitoid medium increased the proportion of larvae entering metamorphosis precociously while injection of antibodies against a parasitoid-released protein had the opposite effect; this indicates that this protein and possibly other parasitoid-released substances are involved in inducing the precocious onset of metamorphosis. Analysis of the plasma proteome of nonparasitized, parasitized and polydnavirus-containing larvae revealed that the developmental effects are associated with only minor differences: eleven low abundant viral or virus-induced proteins and five parasitoid-released proteins were seen at specific stages of the host.

Astrocytes exert a pro-apoptotic effect on neurons in postnatal hippocampal cultures

We investigated conditions that promote basal and activity-dependent neuronal apoptosis in postnatal rat hippocampal cultures. Low-density mixed cultures of astrocytes and neurons exhibited lower sensitivity than high-density cultures to basal neuronal death and activity-sensitive neuronal death, induced with glutamate receptor blockers, sodium channel blockers, or calcium channel blockers. Although elevations of [Ca 2+] i protect neurons from apoptosis, low-density microcultures and mass cultures exhibited only minor differences in resting [Ca 2+] i and Ca 2+ current density, suggesting that these variables are unlikely to explain differences in susceptibility. Astrocytes, rather than neurons, were implicated in the neuronal loss. Several candidate molecules implicated in other astrocyte-dependent neurotoxicity models were excluded, but heat inactivation experiments suggested that a heat-labile factor is critically involved. In sum, our results suggest the surprising result that astrocytes can be negative modulators of neuronal survival during development and when the immature nervous system is challenged with drugs that dampen electrical excitability.

In vitro formation and activity-dependent plasticity of synapses between Helix neurons involved in the neural control of feeding and withdrawal behaviors

Short-term activity-dependent synaptic plasticity has a fundamental role in short-term memory and information processing in the nervous system. Although the neuronal circuitry controlling different behaviors of land snails of the genus Helix has been characterized in some detail, little is known about the activity-dependent plasticity of synapses between identified neurons regulating specific behavioral acts. In order to study homosynaptic activity-dependent plasticity of behaviorally relevant Helix synapses independently of heterosynaptic influences, we sought to reconstruct them in cell culture. To this aim, we first investigated in culture the factors regulating synapse formation between Helix neurons, and then we studied the short-term plasticity of in vitro-reconstructed monosynaptic connections involved in the neural control of salivary secretion and whole-body withdrawal. We found that independently of extrinsic factors, cell–cell interactions are seemingly sufficient to trigger the formation of electrical and chemical synapses, although mostly inappropriate—in their type or association—with respect to the in vivo synaptic connectivity. The presence of ganglia-derived factors in the culture medium was required for the in vitro reestablishment of the appropriate in vivo-like connectivity, by reducing the occurrence of electrical connections and promoting the formation of chemical excitatory synapses, while apparently not influencing the formation of inhibitory connections. These heat-labile factors modulated electrical and chemical synaptogenesis through distinct protein tyrosine kinase signal transduction pathways. Taking advantage of in vitro-reconstructed synapses, we have found that feeding interneuron–efferent neuron synapses and mechanosensory neuron–withdrawal interneuron synapses display multiple forms of short-term enhancement-like facilitation, augmentation and posttetanic potentiation as well as homosynaptic depression. These forms of plasticity are thought to be relevant in the regulation of Helix feeding and withdrawal behaviors by inducing dramatic activity-dependent changes in the strength of input and output synapses of high-order interneurons with a crucial role in the control of Helix behavioral hierarchy.

Insect cellular and chemical limitations to pathogen development: the Colorado potato beetle, the nematode Heterorhabditis marelatus, and its symbiotic bacteria

This research examines possible factors limiting pathogen development and reproduction in a novel host insect. The nematode Heterorhabditis marelatus and its symbiotic bacterium, Photorhabdus luminescens, kill 98% of nematode-treated Colorado potato beetle (CPB) prepupae, but the nematode reproduces in only 1–6% of beetles. We examined nematode/bacterial inhibition at each step of the normal developmental pathway to determine host feature(s) limiting nematode reproduction. We found that in vivo encapsulation of nematodes occurred in only 1.6% of CPB, and in 5% of in vitro hanging drops of hemolymph. Thus, the cellular defense system did not strongly limit nematode reproduction in the CPB. The symbiotic bacterium was negatively affected by a heat-labile factor found in the CPB’s hemolymph which often caused the bacterium to switch from the primary form that produces antibiotics and nutrients necessary for the nematodes’ development, to a secondary form that provides only limited nutrients. A 58 kDa protein was isolated and bioassayed for activity against P. luminescens, but caused a delay in bacterial growth rather than the primary–secondary form switch. Thus, the identity of the heat-labile factor could not be confirmed as being the 58 kDa protein. The heat-labile factor did not directly affect the nematode. The addition of lipids in the form of olive oil to heated CPB hemolymph allowed nematodes to reproduce in 17% of hanging drops, in contrast to zero reproduction in hemolymph without oil. Reproductive nematodes were smaller when grown in CPB hemolymph than in hemolymph of the highly susceptible Galleria mellonella. These data suggest that both the toxic heat-labile factor and a lack of appropriate nutrients alter the CPB–bacterium–nematode interaction. These factors preclude the use of this otherwise highly effective nematode–bacterial complex in the longterm control of the CPB.

Suppression of tumor growth and cell proliferation by p13 II , a mitochondrial protein of human T cell leukemia virus type 1

Human T cell leukemia virus type 1 encodes an "accessory" protein named p13(II) that is targeted to mitochondria and triggers a rapid flux of K(+) and Ca(2+) across the inner membrane. In this study, we investigated the effects of p13(II) on tumorigenicity in vivo and on cell growth in vitro. Results showed that p13(II) significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13(II) exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro. Mixed culture assays revealed that the phenotype of the p13(II) cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13(II) cell lines exhibited an enhanced response to Ca(2+)-mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13(II)-expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13(II) as a negative regulator of cell growth and underscore a link between mitochondria, Ca(2+) signaling, and tumorigenicity.

A Constitutively Active NFATc1 Mutant Induces a Transformed Phenotype in 3T3-L1 Fibroblasts

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is best known for its role in T lymphocyte activation. However, it has become increasingly apparent that this signaling pathway is also involved in the regulation of cell growth and development in a wide variety of different tissues and cell types. Here we have investigated the effects of sustained NFATc1 signaling on the growth and differentiation of the murine 3T3-L1 preadipocyte cell line. Remarkably, we find that expression of a constitutively active NFATc1 mutant (caNFATc1) in these immortalized cells inhibits their differentiation into mature adipocytes and causes them to adopt a transformed cell phenotype, including loss of contact-mediated growth inhibition, reduced serum growth requirements, protection from growth factor withdrawal-induced apoptosis, and formation of colonies in semisolid media. Furthermore, we find that caNFATc1-expressing cells acquire growth factor autonomy and are able to proliferate even in the complete absence of serum. We provide evidence that this growth factor independence is caused by the NFATc1-dependent production of a soluble heat-labile autocrine factor that is capable of promoting the growth and survival of wild type 3T3-L1 cells as well as potently inhibiting their differentiation into mature adipocytes. Finally, we demonstrate that cells expressing caNFATc1 form tumors in nude mice. Taken together, these results indicate that deregulated NFATc1 activity is able to induce the immortalized 3T3-L1 preadipocyte cell line to acquire the well established hallmarks of cellular transformation and thereby provide direct evidence for the oncogenic potential of the NFATc1 transcription factor.