Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene.

The c-fos gene has previously been shown to be transiently induced within minutes after the stimulation of mouse fibroblasts with growth factors. Induction of c-fos was observed specifically with competence factors (e.g., platelet-derived growth factor), not with progression factors (e.g., platelet-poor plasma), suggesting a role for c-fos in conferring competence on fibroblasts. To test this hypothesis we have analyzed c-fos expression in NIH 3T3 cells that were made competent in a different way, namely by wounding a confluent monolayer of cells. Using antibodies raised against either a synthetic fos peptide or a beta-galactosidase--fos fusion protein, we show in this study that in the majority of cells lining the wound c-fos protein is rapidly and transiently induced to high levels. No induction is observed in cells at a distance from the wound greater than approximately 5 cell layers. Induction is equally efficient in both serum-containing and serum-free medium, and is similar in cells that were deprived of fetal calf serum for 40 h prior to making the wound. Our observations support the hypothesis that c-fos may be involved in inducing the 'competent state' in fibroblasts and suggests an early role for c-fos in wound healing and tissue regeneration.     

c-myc and c-fos expression in differentiating mouse primary keratinocytes.

Expression of the myc and fos genes has been monitored in mouse primary keratinocytes after induction of terminal differentiation by calcium or tetradecanoylphorbol acetate (TPA). myc RNA levels in growing cells are very high and remain elevated even at late times after calcium-induced differentiation. Thus, keratinocytes provide the first example of normal primary cells with persistent c-myc expression irrespective of their proliferative or differentiated state. fos expression is also relatively unaffected by addition of calcium. In contrast to calcium, TPA-induced differentiation is accompanied by dramatic changes in proto-oncogene expression: marked c-fos induction and considerable although transient decrease in c-myc expression. These effects might be important for the keratinocyte response to TPA: TPA treatment of a keratinocyte cell line (RBK) resistant to this substance has no effect on c-myc expression and leads only to minimal c-fos induction. In these cells full fos induction can still be triggered by addition of fresh medium. Thus, the fos gene in normal keratinocytes is inducible through at least two independent mechanisms, only one of which has been lost during derivation of the TPA-resistant cell line.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Direct contribution of inducible nitric oxide synthase expression to apoptosis induction in primary smooth chorion trophoblast cells of human fetal membrane tissues

We have previously demonstrated that apoptosis induction is observed only in smooth chorion laeve trophoblast cells, and not in amnion epithelial cells of human fetal membrane tissues prepared at the term. Apoptosis induction was suppressed by the presence of an inhibitor specific for inducible nitric oxide synthase (iNOS), suggesting that intracellular oxidative stress plays a critical role in this process. In this study, we transfected the iNOS gene into primary cultured chorion and amnion cells to examine the direct contribution of iNOS gene expression to the apoptosis induction in these cells. We identified a significant increase in the levels of iNOS protein expression and nitrite accumulation in both chorion and amnion cells after the iNOS gene transfection. However, the induction of apoptosis was observed in an approximately 70% of chorion cells transfected with iNOS gene. Transfection of the iNOS gene into chorion cells resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and downregulation of hemeoxygenase-1 protein expression, whereas no such events were observed in the transfected amnion cells. These results suggest that apoptosis induced in the chorion trophoblast cells by the iNOS gene expression is closely linked to a physiological consequence, such as the rupture of fetal membranes.     

The calcium signal for BALB/MK keratinocyte terminal differentiation counteracts epidermal growth factor (EGF) very early in the EGF-induced proliferative pathway.

BALB/MK mouse epidermal keratinocytes require epidermal growth factor (EGF) for proliferation and terminally differentiate in response to high calcium concentrations. We show that EGF is an extremely potent mitogen, causing BALB/MK cultures to enter the cell cycle in a synchronous manner associated with a greater than 100-fold increase in DNA synthesis. Analysis of the expression of proto-oncogenes which have been reported to be activated during the cascade of events following growth factor stimulation of fibroblasts or lymphoid cells revealed a very rapid but transient 100-fold increase in c-fos RNA but little or no effect on the other proto-oncogenes analyzed. Exposure of EGF-synchronized BALB/MK cells to high levels of calcium was associated with a striking decrease in the early burst of c-fos RNA as well as the subsequent peak of cell DNA synthesis. Since the inhibitory effect of high calcium on c-fos RNA expression was measurable within 30 min, our studies imply that the EGF proliferative and calcium differentiation signals must interact very early in the pathway of EGF-induced proliferation. Our results also establish that c-fos RNA modulation is an important early marker of cell proliferation in epithelial as well as mesenchymal cells.     

Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Arachidonic acid and cyclic adenosine monophosphate stimulation of c-fos expression by a pathway independent of phorbol ester-sensitive protein kinase C

The stimulation of cell proliferation by platelet-derived and other growth factors is associated with a rapid increase in the expression of the c-fos protooncogene. We and others have shown that phosphosphoinositide turnover and protein kinase C play a role in the activation of this gene by growth factors, but that a second, kinase C-independent pathway(s) exist. Because cAMP potentiates the actions of a number of growth factors and is elevated in platelet-derived growth factor-stimulated Swiss 3T3 cells, we examined the ability of cAMP to stimulate c-fos expression in this cell type. Forskolin, a direct activator of adenylate cyclase, elicited marked increases in c-fos mRNA levels. Receptor-mediated activation of adenylate cyclase by prostaglandin E1 and stimulation with the cAMP analog 8-bromo-cAMP also enhanced c-fos expression. In cells made protein-kinase C deficient, c-fos induction by phorbol ester was abolished; by contrast, c-fos was still induced by cAMP-elevating agents in protein kinase C-depleted cells. Platelet-derived growth factor causes cAMP accumulation by stimulating arachidonic acid release and the formation of prostaglandins capable of activating adenylate cyclase. The addition of arachidonic acid and the arachidonate metabolite prostaglandin E2 to Swiss 3T3 cultures stimulated c-fos expression. These data suggest the existence of a pathway from growth factor receptor to gene induction that is mediated by cAMP and does not depend on a phorbol ester-sensitive protein kinase C.     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

The neuroprotective agents chlomethiazole and SB203580 inhibit IL-1beta signalling but not its biosynthesis in rat cortical glial cells

Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen-activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin-1beta (IL-1beta) synthesis, release and signalling in rat cortical glial cells, given the important role of IL-1beta in cerebral ischaemia. We analysed (i) IL-1beta mRNA expression by northern blot, (ii) IL-1beta protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL-1beta protein secreted into the medium by enzyme-linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL-1beta expression by lipopolysaccharide or by IL-1beta itself was very sensitive to nuclear factor kappa B (NF-kappaB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL-1beta-induced expression of c-fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT-PCR, respectively. Because IL-1beta-induced expression of c-fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti-inflammatory properties of chlomethiazole.     

Dynamic characterization of recombinant Chinese hamster ovary cells containing an inducible c-fos promoter GFP expression system as a biomarker.

An inducible reporter gene system for Chinese Hamster Ovary (CHO-DHFR(-)) cells has been developed and characterized with respect to its dynamic properties. The reporter gene system consists of the human c-fos promoter and variants of the green fluorescence protein (GFP), either EGFP with enhanced fluorescence or its destabilized form d2EGFP. The expression of wild-type EGFP or its destabilized form was studied in CHO-DHFR(-) cells in response to serum addition or deprivation. It was shown that serum-induced c-fos promoter mediated EGFP expression was considerably higher than expression from the human CMV promoter, a strong, constitutive promoter preferentially used for high-level expression in CHO cells. However, EGFP was less suitable for studying expression dynamics than d2EGFP due to the protein's long half-life in mammalian cells. The use of d2EGFP resulted in a significant improvement in the dynamic characteristics of the biomarker, particularly when the recombinant cells were selected for high-level GFP expression by subcloning or fluorescence activated cell/sorting (FACS). GFP expression in different subclones and cell populations sorted by FACS was characterized with respect to its dynamic responses in the presence or absence of serum in the culture medium. Significant differences in the GFP expression dynamics were observed for the isolated cell populations. The experimental results indicate that cells with high-level GFP expression also have a faster dynamic response and are thus, desirable for practical application of the reporter gene system e.g. in toxicity monitoring.     

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.