Fos synthesis and neuronal activation: analysis of fos immunoreactivity in identified magnocellular neurons during lactation.
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Animals↗
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Simple non-invasive mapping of pain pathways in living humans, and the effects of acute non-invasively induced pain on substance P, oncogen C-fos Ab1, oncogen C-fos Ab2, dopamine, and acetylcholine.
Most of the present knowledge on pain pathways is based on invasive animal experiments. In 1987, using the Bi-Digital O-Ring Test, Omura found that pain pathways can be evaluated non-invasively in living humans. In this paper, actual examples of such cases are reported. When mild pain was created by pinching different fingers of a normal human subject by placing a plastic clamp on the skin above a pain pathway, the indirect Bi-Digital O-Ring Test, through a fine electroconductive metal wire held by a third person, showed marked weakening of muscle tone of the third person resulting in the opening of the Bi-Digital O-Ring, which was preselected only when the minimum essential requirements to perform the Bi-Digital O-Ring Test were satisfied. The pain pathway thus detected had the following characteristics: For example, when the lateral side of the 5th finger of the left hand was pinched, the pathway was approximately along the ulnar nerve and then went to the dorsal root of the spinal cord around the area corresponding to the lower end of the 7th cervical vertebrae/upper end of the 1st thoracic vertebrae. At this level, the pain pathway goes to the opposite side (right side) then laterally up to the lower one-third of the medulla oblongata. It then turns horizontally from the right side to the left side at the same level, then goes up in the left side of the center of the medulla oblongata. At the left side it goes to the pons, and in the upper part of the pons it turns towards the midline. It then goes up and turns to the right side of the right cerebral cortex corresponding to the lateral side of the 5th finger.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗
From c-fos to v-fos.
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Gene Expression Regulation↗
Free oxygen species (FOS), FOS-scavenging enzyme P-selectin and monocyte activity in cell populations aspirated from early human renal allografts.
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Antibodies, Monoclonal↗
The contribution of NMDA receptor activation to spinal c-Fos expression in a model of inflammatory pain.
1. Intraplantar carrageenin (6 mg 150 microliters-1) evoked a high level of spinal c-Fos expression in the dorsal horn, of segments L4-L5 of the spinal cord, and an extensive peripheral oedema; both parameters were assessed 3 h after carrageenin. 2. Two series of experiments were performed, with the mean total number of Fos like-immunoreactive neurones (Fos-LI), after carrageenin, not being significantly different for the two series of experiments (266 +/- 17 and 332 +/- 31 Fos-LI neurones). For both series of experiments Fos-LI neurones were predominantly located in the superficial and deep laminae, only 10% of the total number of Fos-LI neurones were located in the nucleus proprius and 10% were located in the ventral horn. 3. Pre-administration of the N-methyl-D-aspartate (NMDA) receptor antagonist, (+)-HA966 (0.5 mg kg-1 and 2.5 mg kg-1, s.c.), 30 min before carrageenin, did not significantly influence the total number of Fos-LI neurones, as compared to control carrageenin expression. 4. Pre-administration of the highest dose of (+)-HA966 (10 mg kg-1) significantly reduced the number of deep laminae Fos-LI neurones (28 +/- 3% reduction of control number of Fos-LI neurones after carrageenin, P < or = 0.05), without influencing the number of superficial Fos-LI neurones. There was a tendency towards a reduction of the number of Fos-LI neurones in the nucleus proprius by the highest concentration of pre-administered (+)-HA966, (31 +/- 8% reduction), but this effect did not reach significance. 5. Pre-plus post-administered (+)-HA966 (0.5 mg kg-1), 30 min before and again 45 min afterintraplantar carrageenin, did not significantly influence the total number of Fos-LI neurones, as compared to control carrageenin expression.6. Pre- plus post-administration of 2.5 mg kg-1 (+)-HA966 significantly reduced the total number of Fos-LI neurones, as compared to control carrageenin expression. This effect was reflected by a significant reduction in the number of Fos-LI neurones in the nucleus proprius (36+/-7% reduction of control carrageenin c-Fos expression respectively, P<0.05).7. Pre-plus post-administration of 10 mg kg-1 of (+)-HA966 significantly reduced the number of Fos-LI neurones in the superficial laminae, nucleus proprius, deep laminae and ventral horn (33 +/-0.5%,55+/-6%, 40+/-4% and 51+/-4% reduction of control carrageenin c-Fos expression, respectively, P<0.05,for all areas).8. A single post-administration of (+)-HA966 (10 mg kg-1), 45 min after intraplantar carrageenin, did not significantly influence the number of Fos-LI neurones in the superficial, deep laminae or ventral horn, but significantly reduced the number of Fos-LI neurones in the nucleus proprius, as compared tocontrol carrageenin expression (39+-/8% reduction of control carrageenin c-Fos expression, P <0.05).9 None of the concentrations of (+)-HA966 studied, irrespective of the timing of administration,influenced the peripheral carrageenin oedema. Our results illustrate a contribution of central NMDA receptor activation to carrageenin-evoked spinal c-Fos expression. These results extend previous studies demonstrating the contribution of the NMDA receptor to central hyperalgesia and the expression of c-Fos.
Animals↗
c-fos controls the "private pathway" of light-induced apoptosis of retinal photoreceptors.
White light (5 klux for 2 hr) induces apoptosis of rod photoreceptors in wild-type mice (c-fos(+/+)) within 24 hr, whereas rods of c-fos knock-out mice (c-fos(-/-)) are protected (). The range of this protection was tested by analyzing retinas of c-fos(+/+) and c-fos(-/-) mice up to 10 d after exposure to threefold increased light intensities (15 klux for 2 hr). In c-fos(-/-) mice, rods were unaffected, whereas they were destroyed in c-fos(+/+) mice. After light exposure, mitochondrial damage in rods was observed exclusively in c-fos(+/+) mice. Electroretinograms recorded 48 hr after exposure revealed a decrease of all components in c-fos(+/+) mice but indicated no light-induced loss of function in c-fos(-/-) mice. Thus, in c-fos(-/-) mice, light-induced apoptosis is blocked or its threshold is elevated more than threefold. Increased activity of the transcription factor activator protein-1 (AP-1) in retinas of light-exposed c-fos(+/+) mice indicated an acute contribution of AP-1 to apoptosis induction. AP-1 activity increased already during exposure and peaked approximately 6 hr thereafter, coinciding with the appearance of major morphological signs of apoptosis. Activated AP-1 mainly consisted of c-Fos/Jun heterodimers. In c-fos(-/-) mice, AP-1 activity remained unchanged, indicating that no other Jun- or Fos-family member could substitute for c-Fos. Like damaging light, N-methyl-N-nitrosourea (MNU) induced AP-1 containing c-Fos in c-fos(+/+) mice and did not induce AP-1 in c-fos(-/-) mice. In contrast to light, however, MNU induced apoptosis in rods of c-fos(-/-) mice. Thus, c-Fos is essential for a specific premitochondrial "private apoptotic pathway" induced by light but not for the execution of apoptosis induced by other stimuli.
Alkylating Agents↗
Physiological contributions of neurokinin 1 receptor activation, and interactions with NMDA receptors, to inflammatory-evoked spinal c-Fos expression.
1. Intraplantar injection of formalin (5%, 100 microliters in saline) was associated with a high level of spinal c-Fos immunoreactivity and a peripheral paw and ankle edema, as assessed at 3 h after formalin administration. For the two experimental series, the control number of formalin-evoked Fos-like immunoreactive (Fos-LI) neurons were 174 +/- 6 and 193 +/- 18 (means +/- SE) Fos-LI neurons per 40-microns section of the lumbar segment L4-L5 of the rat spinal cord. For both series of experiments, Fos-LI neurons were located predominantly in the superficial (I-II; 40 and 44% of the total number of Fos-LI neurons for the two experimental series) and deep (V-VI; 37 and 40% of the total number of Fos-LI neurons for the two experimental series) laminae of the dorsal horn of the spinal cord. The small number of remaining Fos-LI neurons were located in the nucleus proprius (laminae III-IV) and the ventral horn. 2. Prior intravenous administration of RP67580 (0.05, 0.5, and 1.5 mg/kg), a selective neurokinin 1 (NK1) receptor antagonist, dose-relatedly reduced the total number of formalin evoked Fos-LI neurons (88 +/- 5%, 80 +/- 4%, P < 0.01 and 64 +/- 4%, P < 0.0001, of the control number of formalin-evoked Fos-LI neurons). Laminar analysis of the regional effect of RP67580 on formalin-evoked Fos-LI neurons illustrated that the number of superficial and deep laminae Fos-LI neurons were attenuated to a similar extent by RP67580. 3. Prior intravenous administration of RP68651 (1.5 mg/kg), the inactive isomer of RP67580, produced only a small reduction in the total number of formalin-evoked Fos-LI neurons (84 +/- 5% of the control number of formalin-evoked Fos-LI neurons (P < 0.05). The effect of RP68651 on the number of formalin-evoked Fos-LI neurons was significantly smaller (P < 0.01) than the effect of the equivalent concentration of RP67580, the active isomer. 4. Prior coadministration of intravenous RP67580 (0.5 mg/kg) and subcutaneous (+)-HA966 (2.5 mg/kg), an antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor, significantly reduced the number of formalin-evoked Fos-LI neurons (64 +/- 4% of the control number of formalin-evoked Fos-LI neurons, P < 0.01). The attenuating effect of coadministered RP67580 and (+)-HA966 was significantly greater than the effect of RP67580 alone (P < 0.01) and the effect of (+)-HA966 alone (P < 0.05). Laminar analysis illustrated that coadministered RP67580 and (+)-HA966 reduced the number of formalin-evoked Fos-LI neurons in the superficial and deep laminae to a similar extent. 5. Intraplantar injection of formalin was associated with a peripheral paw (0.92 +/- 0.02 cm) and ankle (0.92 +/- 0.02 cm) edema, as compared with the paw (0.46 +/- 0.02 cm) and ankle (0.67 +/- 0.14 cm) diameters of saline-stimulated rats. Neither prior administration of intravenous RP67580 (0.05, 0.5, and 1.5 mg/kg) or RP68651 (1.5 mg/kg) or prior coadministration of RP67580) (0.5 mg/kg) and (+)-HA966 (2.5 mg/kg) influenced the extent of the paw or ankle-edema at 3 h after intraplantar injection of formalin. 6. Our results illustrate that NK1-receptor activation contributes to inflammatory-evoked spinal c-Fos expression and thus supports the current contention that NK1-receptor activation, and by inference SP, plays a role in spinal nociceptive processing. The second part of our study suggests that the previously reported NK1/NMDA-receptor interactions contribute to formalin-evoked spinal c-Fos expression and consequently may contribute to the longer term spinal neuroplasticity associated with inflammatory nociceptive processing.
Analgesics↗
c-fos is a positive regulator of carcinogen enhancement of adenovirus transformation.
The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenoviridae↗
Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells.
The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.
Animals↗
The retina of c-fos-/- mice: electrophysiologic, morphologic and biochemical aspects.
PURPOSE: Mice without a functional c-Fos protein (c-fos-/- mice) do not exhibit light-induced apoptotic cell death of rods in contrast to their wild-type littermates (c-fos+/+ mice). To analyze the consequences of the absence of c-fos in the retina, we investigated whether the retinas of c-fos-/- mice have a reduced capacity to absorb and transduce light compared with c-fos+/+ mice. METHODS: Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS: Although the configuration of the ERGs was comparable in both groups of mice, c-fos-/- mice showed a marked variability in all quantitative ERG-measures with lower mean amplitudes, longer latencies, and a 0.9-log-unit lower b-wave sensitivity on average. Morphometry showed that c-fos-/- mice have 23% fewer rods on average, whereas the number of cones was comparable among c-fos+/+ and c-fos-/- mice. Arrestin levels appeared slightly reduced in c-fos-/- mice when compared with c-fos+/+ mice, whereas Hsp70 levels were comparable in both genotypes. The kinetics of rhodopsin regeneration were similar, but c-fos-/- mice had a 25% lower rhodopsin content on average. CONCLUSIONS: Compared with c-fos+/+ mice, retinal function in c-fos-/- mice is attenuated to a variable but marked degree, which may be, at least in part, related to the reduced number of rods and the reduced rhodopsin content. However, c-fos does not appear to be essential for the ability to absorb photons, nor for phototransduction or the function of second-order neurons. The resistance to light-induced apoptosis of photoreceptor cells in c-fos-/- mice may result from the acute deficit of c-fos in the apoptotic cascade rather than from developmental deficits affecting rod photoreceptor function.
Animals↗
Isolation and characterization of a nerve growth factor-regulated Fos kinase from PC12 cells.
Nerve growth factor (NGF) treatment of PC12 cells activates a protein kinase that phosphorylates c-Fos protein at a site near its C terminus, as well as a peptide corresponding to a C-terminal region of c-Fos (Taylor, L. K., Marshak, D. R., and Landreth, G. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 368-372). This serine/threonine kinase, termed Fos kinase, has been purified > 24,000-fold through five column steps to near homogeneity and is shown to be a 37-kDa protein as determined by SDS-polyacrylamide gel electrophoresis (PAGE) with a pI = 6.0. Fos kinase is distinguishable from previously characterized NGF-regulated kinases by its chromatographic behavior, its response to specific kinase inhibitors, and its substrate specificity. The concentration of NGF required to activate Fos kinase is consistent with signaling from the high affinity NGF receptor. Fos kinase phosphorylates c-Fos at its C terminus as indicated by competitive inhibition with a peptide corresponding to C-terminal phosphorylation sites and lack of phosphorylation of a C-terminal deletion mutant of c-Fos. Hyperphosphorylation of c-Fos in vivo, as detected by reduced electrophoretic mobility of c-Fos, is induced by the same ligands which activate Fos kinase. Moreover, Fos kinase phosphorylation of c-Fos in vitro results in a similar electrophoretic mobility shift, demonstrating that Fos kinase may be responsible for growth factor-stimulated alterations in mobility on SDS-PAGE and phosphorylation of this transcription factor. The ability of this unique growth factor-responsive kinase to phosphorylate c-Fos at its C terminus, a region essential for the transrepressive properties of c-Fos, suggests that Fos kinase may play a role in the regulation of the transcriptional repressive activity of c-Fos.
Amino Acid Sequence↗
Cell transformation by c-fos requires an extended period of expression and is independent of the cell cycle.
The proto-oncogene transcription factors Fos and Jun form a heterodimeric complex that binds to DNA and regulates expression of specific target genes. Continuous expression of c-fos causes transformation of cultured fibroblasts and induces osteogenic sarcoma in mice. To investigate the molecular basis of fos-mediated oncogenesis, we developed a conditional cell transformation system in which Fos expression was regulated by isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis or repression of Fos in L1-3c-fos cells occurred rapidly, within 30 min, after the removal or addition of IPTG to the culture medium. However, there was a significant delay between the induction of Fos expression and the appearance of morphological transformation. No effect was observed after 12 h of Fos expression, partial transformation was detected after 24 h, and full transformation required approximately 3 days of continuous Fos expression. Similarly, the transformed cell morphology persisted for at least 2 days after repression of Fos, and a normal phenotype was observed only after 3 days. Fos-Jun complexes, capable of binding to AP-1 sequences, were present continuously during the delay in morphological transformation. Furthermore, increased expression of several candidate Fos target genes, including those encoding Fra-1, transin (stromelysin), collagenase, and ornithine decarboxylase, was detected shortly after Fos induction. The induction of morphological transformation was not dependent on the cell cycle, as it occurred in both cycling and noncycling cells. Thus, the Fos-Jun complexes present before L1-3c-fos cells become fully transformed are transcriptionally active. These complexes disappeared, and the Fos target genes were repressed at least 2 days prior to reversion. Our results suggest that cell transformation by Fos requires increased expression of a target gene(s) with a long-lived product(s) that must reach a critical level.
Animals↗
Role of fos and src in cadmium-induced decreases in bone mineral content in mice.
Cadmium decreases bone mineral in mice and stimulates osteoclast formation and activity in cell culture. Bones from fos-deficient mice contain very few osteoclasts; src-/- bones contain osteoclasts that fail to activate. Fos-/- and src-/- mice develop osteopetrotic bones and their teeth do not erupt. These mice were used to determine if cadmium requires c-Fos or c-Src and secondarily functional osteoclasts to decrease bone mineral content. Mice heterozygous for fos deficiency were mated to produce fos-/- and fos+/o (wild-type) offspring. Pups were divided into four groups: fos+/o, Cd-; fos+/o, Cd+; fos-/-, Cd-; and fos-/-, Cd+. Cd+ pups received daily sc injections (50 microg Cd/kg) on days 17-20 and Cd in drinking water thereafter (10 ppm, days 21-27; 20 ppm, days 27-50). An analogous protocol was followed mating mice heterozygous for src deficiency. For acute exposures, 50-day-old mice were placed on a low-calcium diet and given two sequential 100 microg Cd doses by gavage, and feces were monitored for excretion of bone calcium. Continuous exposure results demonstrate that cadmium (1) significantly decreased bone calcium content (14-15%) and concentration (12-13%) in both fos+/o and fos-/- mice, (2) doubled multinucleated osteoclast-like cell number in fos-/- bones, and (3) stimulated tooth eruption in 40% of fos-/- mice. Cd gavage stimulated bone calcium excretion in both fos+/o and fos-/- mice. In contrast, cadmium had no effect on bones or teeth in src-/- mice. Results indicate that cadmium can decrease bone mineral via a c-Fos-independent pathway; however, c-Src is required for cadmium to stimulate bone remodeling and tooth eruption pathways.
Animals↗
A Fos protein containing the Jun leucine zipper forms a homodimer which binds to the AP1 binding site.
The TPA (12-O-tetradecanoyl-phorbol-13-acetate) responsive element (TRE) is recognized by the inducible transcription factor AP1, a heterodimeric complex of Fos- and Jun-protein subunits, which each contain a specific structure known as the leucine zipper through which they interact. Studies using site-directed mutagenesis have shown that a basic region adjacent to the leucine zipper in Fos is crucial for the interaction of the Fos-Jun complex with the TRE, and probably represents a site of interaction with DNA. The functionally crucial amino acids in this region are almost completely conserved between Fos and Jun (refs 6, 7 and 11; M.N. and R.M., unpublished results), indicating the formation of a nearly symmetrical DNA-binding site in the Fos-Jun complex. Whereas Jun can form a homodimeric protein complex which binds to the TRE, Fos is unable to do so. The Fos-Jun heterodimer, however, possesses at least a 30-fold-higher affinity for the TRE than does the Jun-Jun homodimer, indicating cooperative binding. Because Fos cannot form a homodimer it is not known whether Fos specifically recognizes part of the TRE or has a different role in the binding of the Fos-Jun complex to DNA. Here we report that exchanging the leucine zipper in Fos with that of Jun generates a protein (termed psi-Fos) that can form a complex with Fos. This Fos-psi-Fos complex, and to a lesser extent a homodimeric psi-Fos complex, exhibits specific binding to the TRE. This finding strongly supports the hypothesis that Fos and Jun form a nearly symmetrical DNA-binding site that interacts with the palindromic TRE.
Binding Sites↗
Analysis of c-fos expression in the butyrate-induced F-98 glioma cell differentiation.
The functional induction of c-fos in the sodium butyrate-induced differentiation of F-98 glioma cells was studied. Fos protein level was increased by butyrate. In contrast, c-Jun protein was constitutively expressed and was not affected by butyrate. Gel-retardation assay indicates Fos as a component of the complex formed between the consensus oligonucleotide of the TPA (PMA, phorbol 12-myristate 13-acetate) response element (TRE) and nuclear extract prepared from butyrate-treated cells. Transfection studies showed that butyrate increased transcription from a multimeric TRE-driven reporter construct, and the effect was mimicked by transfecting cells with fos-expression plasmid. Furthermore, under conditions of c-fos over-expression, transactivation by butyrate was essentially abolished. These data suggest that Fos induction had a functional role in gene activation. Characterization of stable c-fos transfectants demonstrated that these cells displayed alterations in morphology, showed serum-dependent growth, had slower growth rates and grew to lower saturation densities than did untransfected F-98 cells or transfected cells that did not express c-fos. Immunofluorescent staining indicated that fos transfectants also had elevated glial fibrillary acidic protein ('GFAP') expression. Transfection of the c-fos promoter-chloramphenicol acetyltransferase fusion gene into F-98 cells revealed that activation of c-fos by butyrate was exerted at the promoter level, and sequences located within nucleotides -757 to -402 of the c-fos promoter were responsible for butyrate induction. Our data indicate that transcriptional activation of c-fos through its promoter by butyrate resulted in increased Fos protein expression. Transfection studies show that both c-fos and butyrate activate TRE-containing genes, and fos may be a downstream mediator of butyrate. Furthermore, expression of c-fos plays a major role in modulating the growth properties of F-98 cells.
Animals↗
DNA binding and transactivation properties of Fos variants with homodimerization capacity.
The mammalian Fos and Fos-related proteins are unable to form homodimers and to bind DNA in the absence of a second protein, like c-Jun for example. In order to study the implications of hydrophobic point mutations in the c-Fox leucine zipper on DNA binding of the entire c-Fos protein, we have constructed and purified a set of Fos mutant proteins harboring one or several isoleucine or leucine residues in the five Fos zipper a positions. We show that a single point mutation in the hydrophobic interface of the c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically to an oligonucleotide duplex harboring the TRE consensus sequence TGA(C/G)TCA. This point mutation (Thr196-->Ile) is situated in the a position of the second heptade (a2) of the Fos zipper. The introduction of additional isoleucine residues in the other a positions progressively increases the DNA binding affinity of these homodimerizing Fos zipper variants. Heterodimerization of these c-Fos variants with c-Jun reveals a complex behavior, in that the DNA binding affinity of these heterodimers does not simply increase with the number of isoleucine side chains in position a. For example, a c-Fos variant harboring a wild-type Thr in position a1 aad Ile in the four other a positions (c-Fos4I) interacts more tightly with c-Jun than a variant harboring Ile in all five a positions (c-Fos5I). The same holds true for the corresponding leucine variants, suggesting that the wild-type a1 residue of the Fox zipper (Thr162) is thermodynamically relevant for Fos-Jun heterodimer formations and DNA binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better than the wild-type zipper protein, suggesting that the driving force for Fos-Jun heterodimerization is not the simple fact that the Fos protein is unable to form homodimers. These c-Fos variants were further tested for their transactivation properties in F9 and NIH3T3 cells. At low expression levels the most efficiently homodimerizing variant (c-Fos5I) activates transcription in F9 cells about 6-fold. However part of this activation may be due to the formation of heterodimers with a member of the Jun family (like JunD for example), since a wild type c-Fos expression vector confers a 3-fold activation under these conditions. In the case of the homodimerizing c-Fos variants however, this activation is abrogated at higher expression levels due to a strong inhibition of basal transcription activity.
3T3 Cells↗
Localization of c-fos-like proteins in the mouse endometrium during the peri-implantation period.
Fos, which collectively refers to protein products of the c-fos proto-oncogene family, was immunohistochemically localized in the mouse uterus to ascertain the effects of steroids on Fos expression in the mouse and to determine the course of Fos expression during normal pregnancy and pseudopregnancy. In ovariectomized mice, Fos was induced in luminal and glandular epithelium and an occasional stromal cell following treatment with 100 ng 17 beta-estradiol (E2). Priming with 2 mg progesterone (P4) for 3 days prior to the administration of 100 ng E2 or giving 100 ng E2 and 2 mg P4 concomitantly induced Fos in the luminal and glandular epithelium with enhanced staining in stromal cells. Priming with P4 before giving E2 had the added effect of inducing Fos at an earlier time point. Progesterone alone failed to induce Fos in any cell type in the uteri of ovariectomized animals. On Days 1 and 2 of pregnancy and pseudopregnancy, Fos was detected in the luminal and glandular epithelium and in the stroma. Staining was decreased by Day 3 and was not detectable by the morning of Day 4. No further staining for Fos was found during subsequent days of pseudopregnancy. On the afternoon of Day 4 of pregnancy, Fos was detected in the epithelium and the antimesometrial stroma. On the morning of Day 5, implantation sites and non-site areas were distinguishable, with Fos present in luminal epithelium and antimesometrial stroma in sites, and in just the luminal epithelium in non-sites. Fos remained in the epithelium of non-sites through Day 7 of pregnancy. In sites on the afternoon of Day 5, staining was found in the luminal epithelium and primary decidual zone. On Day 6, staining was found in the secondary decidual zone, but was absent from the primary zone and epithelium. On Day 7, Fos was also detected in the embryonic trophoblast giant cells and the ectoplacental cone. These results demonstrate that E2 and P4 modulate Fos in the mouse endometrium and that Fos is induced during implantation. Significantly, the embryo appears to induce Fos expression on the afternoon of Day 4, while it is still encased in the zona pellucida.
Amino Acid Sequence↗
Influence of the proto-oncogene c-fos on cisplatin sensitivity.
Cisplatin resistance has been associated with overexpression of the c-fos gene in a human ovarian carcinoma cell line. To determine whether the correlation between c-fos overexpression and cisplatin resistance was limited to this cell line or was a more generalized phenomenon, we investigated cisplatin sensitivity in rat fibroblast cells that overexpressed the c-fos gene. The cisplatin Ic50 values for two different c-fos transfectants, CMVc-fos and L1-3c-fos, were 7.6 +/- 0.8 and 5.6 +/- 1.0 microM, respectively, whereas the cisplatin Ic50 value for the parental line, 208F, was 2.4 +/- 0.1 microM. This represented a 3.2- and 2.3-fold resistance to cisplatin for CMVc-fos and L1-3c-fos cells, respectively. The correlation between c-fos expression and cisplatin resistance also was examined in a human ovarian carcinoma cell line, 2008, and its cisplatin-resistant variant, C13*. Expression of c-fos was elevated slightly at both the mRNA and protein levels in the C13* cells compared with 2008 cells, and c-Fos protein levels were induced in C13* cells following cisplatin treatment. In addition, it was observed that C13* cells were significantly more sensitive than 2008 cells to a c-fos antisense oligonucleotide. The Ic50 values for the c-fos antisense oligonucleotide were 19.9 +/- 5.0 pmol for C13* cells and 58.1 +/- 6.0 pmol for 2008 cells (P = 0.0012). Furthermore, combinations of c-fos antisense and cisplatin reduced the amount of cisplatin required to kill 50% of the C13* cells, although the interaction was not synergistic. These results suggest that expression of the c-fos gene can influence cisplatin sensitivity, and that c-fos antisense oligonucleotide based therapy may be effective at killing parental and cisplatin-resistant ovarian carcinoma cells, either alone or in combination with cisplatin.
Animals↗